4.4 Leafanatomy

BREDENKA1vfP, c.L. & VAN WYK, A.E. 2001a. Leafanatomy ofthe genus . Passerina (Thymelaeaceae): taxonomic and ecological significance. Bothalia 31 : 53­ 70. CONTENTS INTRODUCTION...... 67 MATERIAL AND METHODS ...... 69 Light microscope (LM) studies ...... 69 Scanning electron microscope (SEM) studies ...... 69 Transmission electron microscope (TEM) studies ...... 69 Measurements ofleafin transverse section (tis) ...... 69 Thickness ofthe main vascular bundle ...... 70 Leafwidth ...... 70 Tenninology...... 70 Epidermal structure ...... 70 Cuticle ...... 70 Cuticular ornamentation (LM and SEM) ...... 70 Epicuticular wax ...... " ...... 70 Leafanatomy ...... 70 Crystals ...... 70 Phylogeny ...... 71 RESlJI,TS ...... 71 Macromorphology ofleaf...... 71 Leaf anatomy ...... 71 Trichomes...... 71 Epidermis ...... 71 Tanniniferous substances ...... 72 Crystals ...... 72 Leafstructure, mesophyl1, vascular and sderenchyma tissue in tis ...... 75 Leaf structural types ...... 77 Leaf structural type A ...... 77 Leafstructural type B ...... 77 Leafstructural type C ...... 78

65

Leaf structuraJ type D ...... 78 Comparative leaf anatomy at infrageneric level ...... 78 DISCUSSION AND ADDITIONAL OBSERVATIONS ...... 79 Leaf structure...... 79 Prevailing characters in Thymelaeaceae ...... 79 EpidennaJ tissue ...... 79 Prevailing characters in Tbymelaeaceae ...... 79 Mesophyll ...... 79 Prevailing characters in Thymelaeaceae ...... 79 Ecological significance ...... 81 Leaf structural types: orientation and structure of main vascular bundle in relation to epidennis and mesophyll ...... 81 Prevailing characters ofvascular bundles in Thymelaeaceae...... 81 Xeromorphic gradient ...... 81 EcologicaJ significance ...... 81 Sclerenchyma ...... 82 Prevailing characters in Thymelaeaceae ...... 82 Less important taxonomic characters ...... 82 Leafwidth ...... 82 CrystaJs ...... 82 Tanniniferous substances ...... 82 Phylogenetic considerations ...... 83 Leaf structural type correlated with epidennal structure ...... 83 MesophyU ...... 83 Sclerenchyma...... 83 Taxonomic significance ...... 83 Family level ...... 83 Genus level ...... 83 Species level ...... 83 CONCLUSIONS ...... 84 ACKNOWLEDGEMENTS ...... 84 REFERENCES ...... 84

66 Bothalia 31,1 : 53-70 (2001 )

Leaf anatomy ofthe genus Passerina (Thymelaeaceae): taxonomic and ecological significance

c.L. BREDENKAMP* and A.E. VAN WYK**

Keywords: anatomy, epidermis, mesophyll, Passer;nll, sc1erenchymatous sheath, southern Africa, Thymelaeaceae, vascular bundles

ABSTRACT

A comparative anatomical study was made of the genus Passerina comprising 20 species and four subspecies, most of which are endemic to southern Africa. ]1 showed that anatomical variation is useful in species recognition and classification. Anatomical characters typical of Thymelaeaceae and displayed in Pa.l'serina include isobilatentlleaves, a papillate cuticular membrane, mucilaginous epidermal cell walls, a parenchymatous bundle sheath and extIaxylary sclerenchyma fibres . Vascular bundles of the leaf lack intraxylary phloem. Characters common to Passerina are inverse-dorsi ventral and cpis­ tomatic leaves, inverted palisade parenchyma and an abaxial hypodermal sc1erenchymatous sheath. Orientation of the main vascular bundle in relation to the epidennis and mesophyl\ allows the recognition of four leaf structural types and ten state.~ , according to which all species can be characterized and grouped. Functionally many anatomical features of the leaf in Passerilla are interpreted as adaptations to the Mediterranean climate of the Cape Floristic Region, where most species occur.

CONTENTS INTRODUCTION

Introduction ...... , ...... 53 This paper emanates from a comparative leaf anatomi­ Materi al and methods cal survey of the genus Passerina L.. undertaken as part Light microscope (LM) studies ...... 55 of a monographic study of the group. Work undertaken Scanning electron microscope (SEM) studies .... 55 thus far has revealed at least four new species and four Transmission electron microscope (TEM) studies , 55 new subspecies, to be added to the 16 existing species, of Measurements of leaf in transverse section (tis) . . 55 which most species are endemic to southern Africa Terminology , ...... , ...... , . . . 56 (Thoday 1924; Bond & Goldblatt 1984). Despite the no w Phylogeny ...... ,. 57 outdated revision by Thoday (1924), boundaries of infra­ Results generic taxa in Passerina remain a problem, mainly Macromorphology of leaf ...... 57 owing to the apparent lack of marked morphological dif­ Leaf anatomy ...... , , ...... 57 ferences between the species, This paper explores the Leaf structural types ...... 63 importance of leaf anatomy in Passerina as a source of Key to leaf structural types . , . . ,.,', . " .. ,. 63 potential taxonomic evidence. Leaf structural type A ...... 63 Leaf structural type B ...... 63 Physiography and climate are important in the distri­ Key to subtypes of leaf type B and relevant taxa 63 bution of Passerina (Table I). Most species of Passerina Leaf structural type C . ., ...... 64 are endemic to the Cape Floristic Region. The climate of Key to species ...... , ...... , ...... 64 this region is mostly Mediterranean or semi-Mediterra­ Leaf structural type D .,.,...... 64 nean. Winter rainfall occurs in the west and along the Key to species ...... , ...... , . . .. 64 south coast, complemented by some summer rai n, which Comparative leaf anatomy at infiageneric level .. 64 increases eastwards. The western Karoo and Nama­ Discussion and additional observations qualand (Succulent Karoo Biome) are characterized by Leaf structure ...... , ...... 65 winter precipitation and summer drought. Only two Epidennal tissue .. .. , , ...... , ... , , ... , 65 species, P. sp, nov. 4 and P. montana are distributed to Mesophyll ...... _...... , .. 65 the east and north along the eastern mountains and east­ Leaf structural types: orientation and structure of ern escarpment of southern Africa, areas that receive pre­ main vascular bundle in relation to epidermis dominantly summer rainfall. and mesophyll ...... ,.. 67 Sclerenchyma ...... 68 Less important taxonomic characters ...... 68 Anatomical research in Passerina was initiated by Phylogenetic considerations ..... , .... , ..... , 69 Pick (1882a, b), who studied the effect of light on the ori­ Taxonomic significance ...... ,. 69 entation of assimilation tissue and discussed the inverse­ Conclusions ...... , ...... , .. , .. ... 70 dorsi ventral leaf. Gilg (1891) published a taxonomic Acknowledgements ...... 70 account on the Thymelaeaceae, indicating that the References ...... 70 anatomical structure of stems was similar throughout the family and subsequently of no taxonomic value; he fol ­ lowed Endlicher (1847) in distinguishing the subtribe * National Botanical Institute, Pri vate Bag XIOI, 0001 Pretoria. Passerininae on the basis of floral morphology. Van ** H.G .W.J. Schweickerdt Herbariu m, Depanment of Botany, University of Pretoria, 0002 Pretoria. Tieghem (1893) described the anatomy of the root, stem MS . received: 2000-05-09. and leaf for all the genera of the ThymeJaeaceae known 67 54 Bothalia 31,1 (2001)

TABLE I.-Distribution and habitat of Passerina species (order of taxa according to leaf structural type referred to throughout paper)

Leaf structural Taxon Disuibution Habitat type

A P. sp. nov. I High altitudes, Ceres Karoo Rocky areas, often covered by snow in winter 8 1 P. burchellii High mountains of Genadendal and Villiersdorp South-facing rocky outcrops on mountain summits B I P. pendula KwaZunga Catchment Basin and Zwartkops River On moutain slopes, zone between vaUey bush veld and area of Eastern Cape fynbos, also on plains and roadsides B2 P. comosa Kamiesberg, Great Winterhoelc and Klein Swartberg Mountain slopes and summits Ranges B2 P. paludosa E of Muizenberg Rare in salt marshes on lowland flats B2, B3 P. ga/pinii Agulhas Plain area Calcrete, in stony areas, fields, disturbed areas and roadsides B3 P. drakefUbergUlsis Endemic in Bergvillc District, KwaZulu-Natai Zone between iodigenous forest and alpine grassland, altitude ± I 500-1 980 m. Also a10ng Slreams and river banks B3 P. ericoides Beaches of Western Cape In salt spray, on cliff faces overhanging sea, on primary dunes B3 P. sp. nov. 2 Northern Cederberg Mountains Between large rocks at mountain tops B3 P. sp. nov. 3 Mountain tops of Uitenhage area and Swartberg Pass, In rocky areas and rock crevices Kouga Mountains and Mootagu Pass B4 P. rubra Pon Elizabeth to Uitenhage with outliers in Swellen­ In rocky areas, on f1aner areas, roadsides and disturbed dam and Bredasdorp DiS!. areas B3. B4 P. filiformis L. subsp. Common in Cape Peninsula. distributed from Piquet­ In rocky areas mostly on south-facing mountain slopes fi/ifonnis berg across Hex River Mountains, to AttaquaskJoof in Western Cape B6 P. filiformis L. From Malmesbury NE to Vredendal Deep acid sands subsp. nov. 8 2, B3, P. obtusifolia From Worcester in Western Cape to Alice in Eastern On lower mountain slopes in zone between Karoo B4,B5 Cape. On some mountain ranges in and around Little vegetation and fynbos, otherwise large range of Karoo Karoo habitats, mostly dry rocky areas B2, B3, P. glomerala subsp. Worcester to Tulbagh, in Clanwilliam area, extending Large range of Karoo habitats, mostly dry rocky areas B5 glomerala to Wineberg S of Matjiesfontein B6 P. glomerala From Cederberg Mountains, along mountain summits Rocky crevices on mountain tops and slopes subsp. nov. to Cape Peninsula C P. f alcifolia Mountainous areas between George and Uitenbage On rocky slopes, on mountain passes, roadsides and in flaner field areas C P. sp. nov. 4 From Mossel Bay and Oudtshoom to Eastern Cape In rocky places and rock sheets, also in valleys along and along escarpment northwards to Zimbabwe, with streams outliers in Tanzania B3.82, P. montana Eastern mountains and Great Escarpment of southern In rocky places and rock sheets, also in valleys along DI Africa to Zimbabwe and Malawi streams, altitude ± 1 200 m 82,83, P. paleacea Beaches and salt marshes of Western Cape On primary and secondary dunes as well as rocky 84, D2 areas near sea shore . B3,86, P. rigida Along coast from northern KwaZu1u-Natal to Cape Pioneer and sand binder on primary dunes and rocky D2 Peninsula crevices, common along most of coast D2 P. vulgaris From Western Cape to East London in Eastern Cape In rocky places on mountain slopes and valleys. pioneer in disturbed places, along rouds, even on secondary dunes on coasl at the time and classified the family into three groups, known genera. He classified the famil y into the subfam­ namely Drapetees, Thymelees and Aquilariees, with ilies Aquilarioideae and Daphnoideae and placed Passerina in the Thymelees. In his key, the genera Passerina into the latter, under the tribe Euthymeleae. Lachnaea L., Cryptadenia Meisn., Gnidia L. and Gilg (\894) studied the relationships in the Thyme­ Chymococca Meisn. are grouped on the basis of gela­ Iaeaceae, using mainly fl oral anatomy, and classified the tinized epidermal cells, whereas Passerina is distin­ family into the subfamilies Aquilarioideae, Phaleri­ guished by the absence of these cells. Subsequent work oideae, Thymelaeoideae and Drapetoideae. Passerina is by Bredenkamp & Van Wyk (1999, 2(00) has shown the classified in the subfamily Thymelaeoideae, tribe Daph­ presence of these cells in Passerina. Supprian (1894) neae, subtribe Passerininae. In the same article, he gave studied the stem and leaf anatomy of the Thymelaeaceae, a full account of the anatomical method applied by Va n describing the epidermis ('Hautsystem'), the mechanical Tieghem (1893) and Supprian (1894). concluding that system ('Mechanisches system'), the assimilation system many of the characters used by them were not constant, ('Assimilationsystem'), the vascular structure ('Leitungs­ that there would alw ay,~ be criticism against the anatomi­ system'), the aeration system ('Durchli.ifdtungssystem') cal method and that floral characters were more reliable and excretion management ('ExcretbehaIter') for all the in the delineation of the Thymelaeaceae. 68 BOlhalia 31 , I (2001) 55

Solereder (1899, 1908) summarized the anatomical by adaptations in the leaf structure of the group. The pre­ work on the Thymelaeaceae up to the beginning of the sent paper provides a description of anatomical charac­ twentieth century. The most prominent subsequent ters in Passerina as well as an assessment of their taxo­ anatomical study in the Thymelaeaceae was done by nomic and ecological significance. Leandri (1930), who delimited the Thymelaeoideae on the basis of inlraxylary phloem in the stem. He classified Passerina in the tribe Daphneae, and because of the MATERIAL AND METHODS absence of floral nectaries, into the subtribe 'Passe­ riniinae' , thus combining anatomical and floral charac­ Leaves, both fresh and from herbarium material, of all ters. Hereafter the most signjficant works on the infrageneric taxa in Pas serino were studied. Additional Thymelaeaceae comprised the compilation of aU the Passerina specimens examined since Bredenkamp & available knowledge. Domke (1934) proposed a widely Van Wyk (2000: 70) are listed in Table 2. Leaves from adopted classification system for the family based on herbarium material were rehydrated in water for 5 min­ previous anatomical and floral morphological evidence, utes at boiling point. All leaf material was fixed and classifying Passerina under the subfamily Thyme­ stored in a 0.1 M phosphate-buffered solution at pH 7.4, laeoideae, tribe Gnidieae and subtribe Passerininae. containing 2.5% formaldehyde, 0.1% glutaraldehyde and Finally Metcalfe & Chalk (1950) and Metcalfe (1979, 0.5% caffeine [modified Karnovsky fixative; Karnovsky J983) published accounts of the Thymelaeaceae in their (1965)). Whenever possible, material from at lea,)t five standard works on the anatomy of the dicotyledons. different localities was included.

With the genera in Thymelaeaceae delineated on the Light microscope (LM) studies basis of anatomy and floral morphology, the focus changed to generic revisions. Anatomical work on The light microscope was used for general leaf anato­ Passerino was done by Thoday (1921), who described my and epidermal studies. Unless otherwise stated, the the structure and behaviour of the ericoid leaves of P. fil­ tenth leaf from the growing point of a twig was used in iformis L. and P cr falcifolia C.H.Wright under drought all comparative studies. To prepare semi-thin transverse conditions and supplied some notes on their anatomy. sections, a 1 mm wide segment of leaf material was cut Kugler (1 928) described the inverse-dorsiventral leaves from the centre of each leaf, thus including the main vein ofPfiliformis (= P peetinola Hort.). Recent comparative as well as both leaf margins. Samples were dehydrated, studies on Thymelaeaceae in southern Africa include embedded in glycol methacrylate (GMA) and sectioned those on leaf anatomy of the genera Lachnaea and according to the methods of Feder & O'Brien (1968). Crypradenia (Beyers 1992; Beyers & Van der Walt 1995) Sections were stained in toluidine bl ue '0' , subjected to and on leaf and involucral bract morphology of system­ the periodic acid-Schiff's (PAS) reaction and mounted in atic use in Gn idia (Beaumont et al. 1994). Previous stud­ Entellan (Art. 7961, E. Merck, Darmstadt). ies of leaf anatomy identified mucilagination of the epi­ dermal cells ac; being of possible taxonomic importance. The following three methods were followed in the Recently Bredenkamp & Van Wyk (1999) clarified the study of the cuticles (Bredenkamp & Van Wyk 2(00): structure of these epidermal cells and the origin of the I. GMA sections of leaves in transverse section were mucilage, indicating that this character is of taxonomic stained with I % Sudan Black B dissolved in 70% ethanoL importance mainly at the family level. The present com­ parative anatomical study of the leaves in Passerina is 2. Macerated cuticular mounts were stained with a 1% the most comprehensive to date. Our primary objective aqueous safranin solution. has been to assess the infrageneric taxonomic signifi­ 3. Epidermal mounts, obtained by removing small cance of leaf anatomy in the genus. pieces of ad- and abaxial epidermis manually and by making paradermal hand sections, were stained with 1% The most outstanding anatomical feature of the safranin dissolved in 50% ethanol. Thymelaeaceae, namely the presence of sc!erenchyma fi bres, may well be an adaptation of members of the fam­ Scanning electron microscope (SEM) studies ily to their environments. However, sclerenchyma fibres in the leaves ofPasserina have never been studied at infra­ The scanning electron microscope was used to study generic level before, and this study meets that need. The the epidermal surface features (including epicuticular presence of tough sclerenchyma fibres in the stems of waxes) and to verify the structure of the cuticle (Bre­ Passerina, is well known among indigenous people, who denkamp & Van Wyk 2000). use especially the bark for making ropes, straps and whips.

The wide distribution of Passerina in the Cape Transmission eclectron microscope (TEM) studies Floristic Region, the southern and eastern coastline of South Africa and along the eastern escarpment of south­ The transmission electron microscope was used for ern Africa to Zimbabwe, and with outliers as far north as the study of the structure of mucilaginous epidermal cell Tanzania, provides an opportunity to study the possible walls in Passerina (Bredenkamp & Van Wyk 1999). leaf anatomical adaptations of these plants to a wide range of habitats, experiencing both Mediterranean and Measurements of leaf In transverse section (tis) summer rainfall conditions. For example, we suspect the decreasing rainfall from the eastern escarpment to the Using LM, all measurements were done by using a northwestern parts of the Northern Cape to be refl ected calibrated eyepiece. 69 56 Bothalia 31.1 (200 I)

TABLE 2.-Additional Passerina specimens examined and taxonomical changes made siJJce Bredenkamp & Van Wyk (2000: 70)

Species Voucher specimen Locality

comosa Thoday 212' WESTERN CAPE.-3320 (Montagu): Montagu Dist. . near Concordia, (-CD). filiforrn is subsp.filiformis Boucher 2833' WESTERN CAPE.-31IS (Van RbynsdOlp): Clanwilliam Dis!., Diepkloof S of Verlorevlei, (-AD). Bredenkamp /039 WESTERN CAPE.-331S (Cape Town): Signal Hill, (-(:D). filiformis subsp. nov. Schlechtu 5125' WESTERN CAPE.-32IS (Clanwilliam): Alexander's Hoek, (-BC). Taylor] 542' WESTERN CAPE.- 32IS (Clanwilliam): Malmesbury Div., Vredenburg, Steenberg's Cove, (-CC). glome rata subsp. glomerala Bredenkomp 988" WESTERN CAPE.-32I 9 (Wuppertal): Cederberg Mountains, Eikeboom, (-AC). Bredenkmnp 994 WESTERN CAPE.-3219 (Wuppenal): Cederberg MOWltains, Kromrivier, (-AC). Bredenkmnp J002 WESTERN CAPE.-3219 (Wuppertal): Cederberg Mountains, Algeria, (-AC). Bredenkamp 984, 985 WESTERN CAPE.-3219 (Wuppertal): Citrusdal, Piekeniersldoof Pass, (-CD). Bredenkomp 97T WESTERN CAPE.-3219 (Wuppertal): Groenfontein, (-DC). Bredenkomp 973 WESTERN CAPE.-3319 (Worcester): Tulbagh, (-AC). glome rata subsp. nov. Stokoe 8040' WESTERN CAPE.-3219 (Wuppertal): Cederberg Mountains. Esterlluysen 2858T WESTERN CAPE.-33J9 (Worcester): Hex. River Mounlains. sp. nov. I Goldblall &: Manning 8627 WESTERN CAPE.-3220 (Sutherland): Roggeveld Escarpment, (-AB ). sp.nov.3 Stokoe 9302' WESTERN CAPE.-3322 (Oudtsboom): Swartberg Pass, Prince Albert area, (-AC). Schlechter 584() WESTERN CAPE.-3322 (Oudtshoom): Montagu Pass, (-CD). Esterhuysen 10734' EASTERN CAPE.-3323 (Willowmore): Kouga Mountains, (-DA). Esterhuysen 28006' EASTERN CAPE.-3324 (Steytlerville): Cockscomb, Uitenhage area, (-BD). sp. nov. 4 KUlick 238 KWAZULU-NATAL.-2930 (Piet=narilZburg): Table Mountain, (-CB). Bredenkomp /0/6', JOJ7 ' KWAZULU-NATAL.-3030 (Port Shepstone): Oribi Gorge, (-CB). Van ~k & BredenJwmp 1, KWAZULU-NATAL.-3130 (Pon Edward): Umtamvuna River Bridge, (-AA). 1012'

Bredenkomp 1327 KWAZULU-NATAL.-3l30 (Port Edward): ~kambali Nature Reserve, (-AC). Gillett 4537 WESTERN CAPE.-3422 (Mossel Bay): en route to Knysna from George, (-BB). Keel S.n. WESTERN CAPE.-3423 (Knysna): Knysna, Redlands, (-AA).

•Material used for the SEM study of the 00- and abaxial epidennis. All specimens are housed at PRE.

Thickness of the main vascular bundle Cuticular ornamentation (LM and SEM) The shortest distance between the outermost points of We follow Wilkinson (1979) in our choice of termi­ the ad- and abaxial epidennis through the main vascular nology. bundle, was measured. Epicuticular wax Leafwidth The recognition of soft waxes is based on the criteria Measurements from leaf margin to leaf margin would proposed by AmeJunxen et al. (] 967). Further interpreta­ be unreliable considering tbe cymbiform sbape of the tion of epicuticular waxes and crystals was done accord­ leaf, the involute nature of the leaf margins, and the fact ing to Wilkinson (1979) and BartWott et al. (1998 ). that leaf margins correspond to changes in turgor pres­ sure. Leaf width was therefore measured as the shortest Leaf anatomy distance between the outer epidermal walls of the abaxi­ The definitions of dorsi ventral and isobi lateral leaves al epidermis, parallel to the adaxial epidennis, but per­ (Eseau 1965; Metcalfe 1979; Fahn 1982; Mauseth 1988) pendicular to the axis of the main vascular bundle. are accepted in the present study. However, we follow Kugler (1928) who coined the term 'inverse-dorsiven­ Terminology tral' for leaves where the palisade parenchyma develops abaxialJy, because the abaxial epidermis is exposed to Epidermal structure the environment. In addition to leaf symmetry, the works of the above-mentioned authors as well as those of Sole­ Epidermal structure was described by Bredenkamp & reder (1908) and Metcalfe & Chalk (1 950) have been Van Wyk (1999, 2000). used for the interpretation of leaf anatomy.

Cuticle Crystals Following l effree (1986), we distinguish the cuticle Crystals were interpreted according to Metcalfe proper, the cuticular layer and the cell walL (1983). Unstained GMA sections of leaves were used to 70 57 Bothalia 31,1 (2001)

identify the presence and position of crystals by means of late. Leafbase sessile or cuneate. Leaf apex truncate and polarized light, after which they were tested for the pres­ hump-backed, obtuse, rounded, acuminate or acute to al­ ence of flavonoid glycosides, such as diosmin. according most spine-tipped. Margins sometimes ciliate. Size (1.5-) to the method of Jackson & Snowdon (1990). The iden­ 2.5-4.0(-8) X (0.8-)1.2-2.0(-3.0) mm. Figure I. tity of the crystals was fmally confirmed using energy dispersive spectrometry (EDS). Leaf anatomy Phylogeny Trichomes Speculations on phylogeny are based on the prevail­ Adaxial surface of leaf villous, with uniseriate tri­ ing family characters representing the ancestral state and chomes forming a felty layer over adaxial epidennis; tri­ derived characters at genus and species levels considered chomes bordering leaf margin often conspicuous. as possibly advanced. A cladistic analysis based on Abaxial surface of leaves mostly glabrous, young leaves anatomical and morphological characters in Passerina is tomentose to sparsely hairy in a few species (Table 3; scheduled for the fi nal stages of the study. Figure 2A).

RESULTS Epidermis

Macromorphology of leaf lAmina epistomatic. AdLuial epidermis uniseriate, following concave leaf surface; stomata anomocytic; Leaf arrangement decussate, sometimes imbricate, cuticular membrane 2-5 ~ m thick; epidermal cells irreg­ closely adpressed to stem or spreading at an angle of ularly shaped, outer periciinal wall convex in tis, polyg­ 5°_20°(-60°); spreading of leaves often prominent in onal in surface view. Abaxial epidermis uniseriate, con­ juvenile plant'>. lAmina inversely ericoid; adaxial surface vex (following leaf surface curvature). glabrous or concave. villous, often forming a groove facing the stem; sparsely hairy; cuticular membrane well developed, abaxial surface convex, orientated more or less acroscop­ (10-)20-30(- 70) ~m thick in tis; epicuticular waxes ically, thus exposing a large surface area to the environ­ coating ,entire surface, crystalloids, wax platelets and ment; cuticle often amber-coloured (in herbarium materi­ plates present or absent (Figure 2B-D). Anatomical al) and outline of epidermal cells often macroscopically structure and taxonomic value of abaxial epidermis are visible. Leaf shape cymbifonn. falcate or cigar-shaped; discussed by Bredenkamp & Van Wyk (2000), and cor­ plane shape linear. oblong. lanceolate, or narrowly trul­ related with leaf structural types in Table 3.

TABLE 3.-Correlation between leaf structural type and epidenna! characters (Bredenkamp & Van Wyk 2(00) in Passerina

Leaves Abaxial Epiden:nal cells Ornamentation hair of Taxon lsobilat- Inverse­ arranged arranged cuticular membrane era! dorsi ventral rnndomly in rows Type A TypeB TypeC TypeD Group A Group B Smooth Papillate Striate

P. sp. nov. ! (Figure 3A) A X X P. burcile";i B I X X P. peruJula (Figure 3B) Bl X X P. comosa 82 X X X P. paludosa (Figure 3C) B2 X X P. galpinii (Figure 2C) B2. B3 X X P. drakensbergensis B3 X X P. ericoides (Figure 3D) B3 X X P. sp. nov. 2 B3 X X P. sp. nov. 3 83 X X X X P. rubra 84 X X P. jili/ormis sUbsp.jili/ormis B3, B4 X X (Figure 3E) P. jiliformis subsp. nov. B4, 86 X X P. obtusifolia (Figure 3G) B2, B3, X X B4,B5 P. glome rata subsp. g/omerata B2, B3, X X (Figure 3F) B5 P. glomeraJa subsp. nov. 86 X X P.falcifolia (Figure 4A) C X X P. sp. no v. 4 (Figure 4B) C X X P. montana (Figure 4C) B2, B3 Dl X X X P. paleacea (Figure 4D) B2, 8 3, D2 X X 8 4 P. rigida (Figures 3H, 4E) B3, 86 D2 X X P. vulgaris (Figure 4F) D2 X X

71 58 Somalia 31.1 (200 I)

..~ A B :.. c o - ....•.. ~ {l. .

E F H

lea' tissue

M N

/~ I a) . ~ - ~. '- ~ Q R s T

FIGURE I.-Camera lucida drawings of leaves of Passerillo species arranged according to leaf slrUcrural rype (Table 3). cOITclating leaf shape [() outline of leaves in Us: A. P. sp. nov. I, Bredenlwmp 1044; B, P. hurchellii. Bolus 684; C, P. pendula, lJredellkamp 90B; D. P. ('(lIlIO.\'({, Andreae 1288; E, P. paludosa. B1l!denlUlmp 1035; F. P. ga/pinii. Bredenkalllp 946; G. P. drakellsberge1lsis, Brede/llwmp 1019; H. P. eri­ coides. Bredenlwmp 962; I, P. sp. nov. 2. Esterhllysen 26859; J, P. sp. nov 3, ESlerhllysen 28006; K. P. mbra, Bredenkamp 914: L, P. fili­ jomlis subsp. fi/ijomlis, Bredenkamp 896; M, P. oblllsijolia, Bredenlwmp 919; N, P. glomerata subsp. glomerata, Bredel1kamp 984; 0, P. jalcijolia, Bredenkamp 917; P, P. sp. nov. 4, Bredenlwmp 1016: Q, P. "umlana, Bredenkamp 889; R, P. paleacca, Bredenkal1lp 960: S, P. rigida, Bredenk(lll1jl 911; T, P. vulgaris, Bredenkamp 901 . Leaf size A-T x 20. Scale bar: 10 mm; leaves in Us not to scale.

Tannin!ferous substances Crystals Tanniniferous substances (Figure 2C, D) present in Calcium oxalate crystaJs (Figure 2E, F) present in mes­ ad- and abaxial epidennis, mesophyU, bundle sheaths, ophyJl, clustered crystals (druses) jn parenchyma, frag­ parenchyma abaxial of vascular tissue in vascular bun­ mented calcium oxalate crystals, resembling crystal sand, dle, staining homogenously or with vesicular appear­ present in intercellular spaces; flavonoid glycosides (dios­ ance. min) absent. 72

Bothalia 31,1 (2001) 59

FIGURE 2.- A, B, SEM micrographs of trichomes and epicuticular waxes: A, P.faleifolia, Bredenkamp 915; B, P. rigida, Bredenkamp 1013. C-H, LM photographs of leaf: C, P. galpinii, Bredenkamp 946; D, P. pendula, Bredenkamp 909, stomatal apparatus in adaxial epidermis stained with Sudan Black B; E, P. paleacea, Pillans 3783, calcium oxalate crystals and druses in polarized light; F, P.faleifolia, Tyson 1449, cluster crystal from ruptured palisade parenchyma in polarized light; G, P. drake.nsbergensis, Bredenkamp 1019, mesophyll in leaf margin; H, P. glome rata subsp. glomerata, Bredenkamp 977, mesophyll and tracheid in leaf margin. ab, abaxial epidermis; ad, adaxial epidermis; bs, bun­ dle sheath; c, cambial cells; CM, cuticular membrane; co, calcium oxalate crystals; cy, collenchyma; d, druse; e, epidermal cell; ef, extraxy­ Jary sclerenchyma fibres; gc, guard cell; ii, inner ledge; m, mucilage; mb, median vascular bundle; 01, outer ledge; ph, phloem; pi, plates; pp, palisade parenchyma; pr, peristomatal rim; ps, platelets; s, sclerenchymatous hypodermal sheath; sb, secondary vascular bundle; sp, spongy parenchyma; st, stomata; t, trichome; ta, tanniniferous compounds; tr, tracheid; X, xylem. Scale bars: A, B, D, 10 J.lm; C, E-H, 100 flm. 73

60 Bothalia 31,1 (2001)

-

FIGURE 3.-LM photographs of leaf structural types A and B. Type A: A, P. sp. nov. 1, Bredenkamp 1044. Type B I: B, P. pendula, Bredenkamp 908. Type B2: C, P. paludosa, Bredenkamp 1035. Type B3: D, P. ericoides, Bredenkamp 962. Type B4: E, P.filifonnis sUbsp.filiformis, Bredenkamp 1039. Type B5: F, P. glome rata subsp. glomera/a, Bredenkamp 984, with secondary tissue in vascular bundle; G, P. obtusi­ folia, Bredenkamp 919. Type B6: H, P. rigida, Bredenkamp 1013. Abbreviations as for Figure 2. Scale bars: A-H, 100 flID.

74 Bothalia 31,1 (2001) 61

FIGURE 4.-LM photographs showing leaf structural types C and D. Type C: A, P.falci/olia, Bredenkamp 917; B, P sp. nov. 4, Bredenkamp 1016. Type DI: C, P. montana, Bredenkamp 889. Type D2: D, P. paleacea, Bredenkamp 960; E, P. rigida, Bredenkamp 962; F, P. vul­ garis, Bredenkamp 901. Abbreviations as for Figure 2. Scale bars: A-F, 100 flm.

Leaf structure, mes9phyll, vascular and sclerenchyma cymbiform leaf folding along reinforced midrib; thick­ tissue in tis ness of main vein (260-)440(-560) flm. Mesophyll pal­ isade-like and homogeneous or inverted-spongy Leaf isobilateral or inverse-dorsi ventral. Outline parenchyma situated adaxially and palisade parenchyma variable, narrowly transversely elliptic in flatter leaves, abaxially. Palisade parenchyma horseshoe-, U- or V­ transversely elliptic (Figures 1F; 3A; 4C, E) or trans­ shaped, 1- or 2-layered, or 2- or 3-layered, (3)4 or 5(6) versely oblong (Figure 1R) in cymbiform leaves, also cells per 50 11m; cells narrowly elliptic to elliptic in iso­ canaliculate (Figures 1G; 3H) or carinate (Figures bilateral leaves or elongated in inverse-dorsi ventral 10-P; 4A, B, F); adaxial epidermis slightly concave in leaves, containing chloroplasts, tanniniferous deposits flatter leaves or leaf lamina and margins strongly and druse crystals. Spongy parenchyma in isobilateral upturned to involute forming a central groove or fur­ leaves ± uniform in shape, cells narrowly elliptic to row. Margins filled with palisade parenchyma extend­ elliptic, densely arranged with larger intercellular ing to adaxial epidermis, abruptly becoming irregular spaces in centre of leaf, aerenchymatic, meso- or xero­ adaxially, conforming to shape and size of spongy morphic in inverse-dorsi ventral leaves, often resem­ parenchyma (Figure 2G); terminal vein endings often bling palisade parenchyma adaxial to veins, rounded, present, ultimately consisting of a single tracheid pentagonal or heptagonal with lobes connecting neigh­ (Figure 2H). Width (570-)880(-1480) 11m. Midrib often bouring cells; cells either loosely arranged with large raised below owing to supporting sc1erenchyma fibres, intercellular spaces or densely arranged with small,

75 Bothalia 31, I (200I) 63

obovate. Bundle sheath completely enveloping vascular tral leaves (Figure 5). In this transformation series, bundle (Figure 3A-H) or present adaxially only (Figure fo lll' leaf structlll'al types and ten states are identified 4A-F); number of cells (1 2- )14-28(- 32), mostly ODe (Tables 3; 4). layer of parenchymatous cells or irregularly two-lay­ ered; cells larger than other tissues in vascular bundle. roundish or often longitudinally or transversely elon­ Key to leaf structural types gated; tanniniferous deposits ample. Vascular tissue collateral with adaxial xylem and abaxial phloem, sur­ Ia Leaf isobilateral _ . . . . . __ . __ _ . type A (Figures 3A; SA) I b Leaf ioverse-dorsiventra.l: rounded by a layer of colourless parenchymatous cells, 2a Bundle sbeadt completely enveloping main vascular bun­ becoming tanniniferous or sclerenchymatous abaxially, dle, extraxylaI}' sclerenchyma fibres enclosed in bun­ separating vascular tjssue from abaxial sc1erenchyma dle shearh ...... type B (Figures 3B-H; 5B) fibres. Xylem arranged in a ± semilunar band or in ± 7 2b Bundle sheath capping main vascular bundle adaxially, ± radial ti ers, alternating with xylem parenchyma absent abaxially, extraxylary sderenchyma fibres not enclosed in bundle sheath: (Figures 2C; 3F-G; 4A, F), often separated from 3a Main vascular bundle bordering on palisade parenchy­ phloem by thin-walled parenchyma cells. Phloem ma, eXUlIXylary sclerenchyma fi bres fitting into V­ arranged in shallow band, sieve tubes and companion shaped palisade parenchyma ... type C (Figures 4A, B; 5C) cells interspersed with phloem parenchyma. Secondary 3b Main vascular bundle extending beyond palisade paren­ chyma, extraxylary sclerenchyma fibres in contact growth indicated by cambial cells and thin-walled with abaxial epidennis: derivatives arranged in rows between secondary xylem 4a Sclerenchymatous hypodermal sheath absent __ ... .. and secondary phloem; primary xylem situated in most _. _ ...... type D I (Figures 4C; 5D) adaxial position between large parenchymatous cells 4b Sclerenchymatous hypodermal sheath present ...... and primary phloem bordering on sclerenchyma fibres, ...... type D2 (Figures 4D-F; 5D) conspicuous in P. glome rata subsp. glomerata (Figure 3F). Extraxylary fibres bordering phloem tissue abaxi­ Leaf structural type A ally, ± separated by irregular layer of parenchyma often Leaf isobilateral. Main vascular bundle central; bun­ containing tanniniferous substances and becoming scle­ dle sheath completely enclosing vascular tissue. Second­ renchymatous (Figures 2C; 3F, H); enclosed in bundle ary vascular bundles close to one another, with bundle sheath or extending beyond bundle sheath and palisade sheaths adhering. fonning a central plate of veins. parenchyma up to abaxial epidennis. Secondary vascu­ Sclerenchymatous hypodermal sheath absent (Tables 3; lar bundles (5)6-13(- 19) corresponding to main vascu­ 4; Figures 3A; SA). Species represented: P. sp. nov. I lar bundles in orientation, shape, bundle sheath and (Figure 3A). arrangement of vascular tissue. Sclerenchymatous hypodermal sheath formed by sclerenchyma fibres Leaf structural type B extending paradermally. often up to leaf margins ('wan­ dering' fibres sensu Tboday 1921), connecting with Leaf inverse-dorsi ventral. Palisade parenchyma fibres from main and secondary vascular bundles in horseshoe-shaped. Main vascular bundle variously orien­ P. paleacea, P rig itio , and P vulgaris (Figures 4D-F; tated in relation to mesophyll: adhering to adaxial epider­ 5); sclerenchyma fibres in mesophyU (,Spicularzellen' mis (type BI), centrally arranged with strands of spongy sensu Supprian 1894) absent. parenchyma (type B2), touching palisade parenchyma abaxially (type B3), sunken into palisade parenchyma (type B4), extending beyond palisade parenchyma with Leaf structural types collenchyma wedged between main vascular bundle and abaxial epidermis (type B5) or adhering to abaxial epi­ The isobilateral and inverse-dorsi ventral states of dermis (type B6). Shape ovate, widely ovate, very wide­ the leaf dictate the orientation of the main vascular ly ovate, oblate or obovate. Bundle sheath completely en­ bundle. The main vascular bundle is central in isobilat­ veloping main and secondary vascular bundles. Extra­ eral leaves (Figure 3A). and either close to the adaxial xylary scierenchyrrUl fibres enclosed in bundle sheath. epidermis. situated oentrally, or variously arranged in Sclerenchymatous hypodermal sheath absent (Tables 3; 4; relation to the abaxial epidermis in inverse-dorsiven­ Figures 3B- H; 5B).

Key to SUbtypes of leaf type B and relevant taxa

I a Main vascular bundle close to or adhering to adaxial epidermis ...... type B 1: P. burchellii, P. pendula (Figure 3B) 1 b Main vascul ar bundle central or abaxial: 2a Main vascular bundle central .. . type 82: 3a Mesophyll inverse-dorsiventral, spongy parenchyma more homogenous and palisade-li ke: 4a Main vascular bundle surrounded by aerenchymatic spongy parenchyma, strands of spongy parenchyma connect­ ing main vascular bundle 10 adaxial epidermis, palisade parenchyma and secoodary vascular bundles ...... _...... P. paludosa (Figure 3C) 4b Main vascular bundle surrounded by mesomorphic spongy parenchyma, secondary vascular bundles closely arranged ...... _ ...... P. comosa 3b Mesophyll inverse-dorsi ventral. palisade and spongy parenchyma clearly di stinguished: Sa Mesopby\l xeromorphic ...... _...... P. oblusijolia, P. gioml!rara subsp. glomerala 5b Mesopbyll aerenchymatic or mesomorphic: 6a Oulline of leaf in tis transversely elliptic ...... __ ...... P. galpinii (Figure 2C), P. monraTUl (Figure lQ) 6b Outline of leaf in tis transversely oblong...... P. paleacea (Figure I R) 2b Main vascular bundle abaxially arranged or interfering with palisade parenchyma: 77 64 Sothalia 31.1 (200 I)

7a Main vascular bundle touching palisade parenchyma abaxially ... type B3: 8a Abaxial epidennal cells large. srrongly mucilaginous. peridinal x anticlinal dimensions in tis 30-65 x (-35)55-60 (-70) !lm: 9a Palisade parenchyma V-shaped ...... P. paleacea 9b Palisade parenchyma borseshoe-shaped ... P. gaJpinii, P drolc£nsbergensis, P ericoides (Figure 3D), P. rigida, P. montana 8b Abaxial epidermal cells exceptionally large. abundantly tanniniferous, mucilagination minimal, periclinal x anti­ clinal dimensions in tis (20-)30-45(-50) x (25-)30-75(-105) )lm: lOa Outline of leaf in tis transversely elliptic to cordifonn ...... P. obtusifolia (Figure 1M). P. glomerata subsp. glomerata (Figure IN), P.filiformis sUbsp.fili/ormis (Figures I L) lOb Outline of leaf in tis not transversely elliptic, depressed obovate or canaliculate respectively ...... P. sp. nov. 2 (Figure 11), P. sp. nov. 3 (Figure IJ) 7b Main vascular bundle sunJc:en into palisade parenchyma, causing specialization of, or extending beyond palisade parenchyma: Ila Palisade parenchyma indented because of sunken main vascular bundle ... type B4: 12a Abaxial epidennal cells srrongly mucilaginous, periclinal x anticlinal diam. in tis 30-65 x 45-60 flm: 13a Leaf outline in tis canaliculate; secondary vascular bundles ± 6 on each side of main bundle .. . P. rubra (Figure I K) 13b Leaf outline in tis transversely oblong, margins erect; secondary vascular bundles 3 or4 on each side of main bundle...... P. paleacea (Figure IR) 12b Abaxial epidermal cells strongly tannioiferous, periclinal x anticlinal cell diam. in tis (35-)40-45 x 45-75 (-105)!lm ...... P.filifonnis sUbsp.filiformis (Figure 3E), P. filiformis subsp. nov., P. obtusifolia II b Palisade parenchyma abaxial of main vascular bundle specialized, or main va'iCular bundle extending beyond pal­ isade parenchyma: 14a Main va~cular bundle separated from abaxial epidennis by collenchyma containing ample amounts of tanninife­ rous substances...... type B5: P. glomerala subsp. glomerala (Figure 3F), P. obtusifolia (Figure 3G) 14b Main vascular bundle extending beyond palisade parenchyma, bordering on abaxial epidermis, abaxial cells of vascular bundle sheath often collenchymatous ... type B6: 15a Main vascular buodle abaxially orientated, shape ovate 10 very widely ovate ...... P.filiformu subsp. nov., P. glome rata subsp. nov. 15b Main vascular bundle exceptionally large, situated close to adaxial epidermis, reaching and touching abaxial epidermis, shape elliptic ...... P. rigida (Figure 3H)

Leafstructural type C structural type D2, absent in P. montana (type 01) (Figures 4C- F; 5D). Leaf inverse-dorsiventral. Palisade parenchyma V­ shaped. Main vascular bundle bordering on palisade Key to species parenchyma abaxiaJly; shape obovate; bundle sheath I or 2 layers of parenchymatous cells capping main vascular I a Sclerenchymatous hypodennal fibres absent .... bundle adaxially, ± absent abaxially; cells rounded or ...... type 01: P. montana (Figure 4C) longitudinally lobed, containing ample amounts of tan­ I b Sc1erenchymalous hypodermal fibres present .. . type 02: niniferous substances. Extraxylary sclerenchyma fibres 2a Outline of leaf in tis transversely oblong; parts of lamina extending beyond adaxial epidennjs narrow, contain­ not enclosed in bundle sheath, bordering on and fitting ing tenninal vascular tissue only; margins uprurned: into the V-shaped palisade parenchyma. Sclerenchyma­ central furrow wide and shallow; palisade parenchy­ tous hypodermal sheath absent (Figures 4A, B; 5C). ma V-shaped, density 5 cells per 50)lm ...... P. paleacea (Figures I R; 40) 2b Outline of leaf in tis transversely elliptic or carinate; parts Key to species of lamina extending beyond adaXial epidermis varying in width, each containing (2)3(-6) secondary vascular la Bundle sheath cells longitudinally lobed. radiating outwards bundles; margins diverging outward or involute; cen­ adaxially, containing ample amounts of tanniniferous tral groove deep; palisade parenchyma horseshoe- or substances...... P. ja/cifo/ia (Figure 4A) V-shaped, density (3)4(5) cells per 50 )lm: I b Bundle sheath cells rounded ...... P. sp. nov. 4 (Figure 4B) 3a Outline of leaf in tis transversely elliptic; margins invo­ lute; palisade parenchyma horseshoe-shaped . . . '...... P. rigida (Figure 4E), less often P. vulgaris 3b Outline ofleaf in tis carinate; margins diverging outward Leafstructural type D or involute; palisade parenchyma V-shaped ...... 20. P. vulgaris (Figure 4F) Leaf in verse-dorsiventral. Palisade parenchyma horseshoe-, U- or V-shaped. Main vascular bundle Intermediate states, with main vascular bundle orien­ extending beyond palisade parenchyma, ultimately in tated in more than one position in relation to the ad- and contact with abaxial epidermis. Shape obovate. Bundle abaxial epidermis and mesophyll, were recorded in sheath I or 2 layers of parenchymatous cells capping P. glomerata subsp. glomerata, P. obtusifolia, P. montana, main vascular bundle adaxially, ± absent abaxially; cells P. paleacea and P. rigida. All states were accounted for in rounded or lobed. Extraxylary scierenchyma fibres Tables 3 and 4, as well as in the construction of keys. extending beyond bundle sheath, running through pal­ isade parenchyma up to abaxial epidennis; not enclosed in bundle sheath (ultimate stage in P. montana, type Dl). Comparative leaf anatomy at infrageneric level Sclerenchymatous hypodermal sheath formed by hypo­ dermal fibres extending paradermally in direction of leaf In order to facilitate the interpretation of existing data margins, often connecting with fibres from main and sec­ for all infrageneric taxa, data are summarized in Tables 3 ondary vascular bundles. Present in all species of leaf and 4. 78 Bothalia 31, J (2001) 65

DISCUSSION AND ADDITIONAL OBSERVATIONS mal characters being more affected by environmental change. However, the arrangement of the epidermal Leaf structure cells and the ornamentation of the cuticular membrane (CM) correlate well with leaf structural type (Table 3). Prevailing characters in Thymelaeaceae Species with epidermal cells arranged randomly and with smooth or papillate cuticular ornamentation, all Leaf structure in Thymelaeaceae exhibits a transfor­ have leaf structural type B. With the exception of P. mation series from mainly dorsiventral. the prevailing montana (type Dl) with epidermal cells arranged in state in the family, to isobilateral or centric in Diarthron rows and smooth or papillate cuticular ornamentation, Turcz., Pimelea Banks & Sol. and Thymelaea Juss. structural types C and D correlate with epidermal cells (Leandri 1930; Metcalfe & Chalk 1950), al1 these states arranged in rows with striate cuticular ornamentation. being present in Lachnaea and Cryptadenia (Beyers Finally, structural type D is also characterized by a 1992; Beyers & Van der Walt 1995 ) and the inverse-dor­ well-developed sclerenchymatous hypodermal sheath siventraJ state prominent in Passerina. Thoday (1921) (Table 4). possibly representing the more advanced recorded the isobilateral state in juvenile leaves in select­ state. ed species of Passerina, possibly reflecting its primitive status in the genus. Leaves of P. sp. nov. I (Figures 3A; 5A) are isobilateral. whereas the leaves of all the other MesophyD species are inverse-dorsi ventral. possibly representing the more advanced state (Figures 3B- H; 4A- F; 5B- D). Prevailing characters in Thymelaeaceae

Most species of Passerina are adapted to the dry, Palisade cells are generally short. Mesophyll. includ­ wann summers and humid winters of the winter rainfall ing irregular sclerenchymatous fibres, is found in species area of the Cape Floristic Region, olhers survive in the of Daphne L., Daphnopsis C.Mart., Enkleia Griff., arid conditions of the Karoo, some grow in a range of Gyrinops Gaertn., Peddiea Harv. and Stephanodaphne habitats along the eastern escarpment and some are Baill. (Metcalfe & Chalk 1950). adapted to maritime conditions along the southern African coast (Table 1). What appears to be adaptations In the isobilateral leaf of P. sp. nov. 1, mesophy\l is to these varying environments are reflected in the leaf palisade-like and homogeneous. All other species of structure of the various species. Weigljn & Winter Passerina display the possibly more advanced state in (1991), studying the morphological-anatomical features which the mesophyll is inverted, with elongated, abaxial of perennial halophytes, pointed out the importance of palisade parenchyma and adaxial spongy parenchyma. curvature of outer epidermal cell walls, epicuticular The orientation of the mesophyU in rel ation to leaf shape, waxes, mesophyll orientation, enrolled leaves and forti­ sclerenchyma and vascular tissue (Table 4), forms the fication of tissue--characters present in most Passerina basis of various leaf structural types distinguished in this species. Other important leaf adaptations in Passerina study. The palisade parenchyma is horseshoe-shaped in are the decussate and appressed arrangement. the cymb­ all species with narrowly transversely elliptic. cordiform iform shape and the inverted palisade parenchyma. or canaliculate leaves in tis and with leaf structural type developing on th at side of the leaf which is exposed to B (Figure 3B-H). In P. paleacea, the leaf is transversely the highest light intensity. Structural support is ren­ oblong in tis and the palisade parenchyma is U-shaped dered by the presence of scIerenchyma in the main and (Figures I R; 4D). Palisade parenchyma is V-shaped secondary vascular bundles and in some Western Cape (Figure 4A, B, F) in species with structural types C and species these fibres proliferate beyond the vascular D, in which the leaves are typically carinate in tis. In the bun'dle sheath to join with hypodennal fibres. forming most xeromorpic state, sclerenchyma extends through a sclerenchymatous hypodermal sheath. The impor­ the V-shaped palisade parenchyma, joining other hypo­ tance of many of these characters is further discussed dermal fibres to form a sclerenchymatous hypodermal below. sheath (Figure 4F). Irregular sclerenchymatous fibres are absent in the mesophyll of leaves in Passerin<1 .

The mesophyll of the leaf is seemingly adapted to sur­ Epidermal tissue vive arid conditions and high light intensity, thus becom­ ing xeromorphic in most spec ies. This is reflected by the Prevailing characters in Thymelaeaceae palisade parenchyma which usually occurs in 1-3 layers, qui te densely arranged with 3-6 cell s per 50 ~ m . According to Metcalfe & Chalk (1950) the cuticular Palisade cells contain large numbers of chloroplasts, membrane (CM) of the leaf is usually smooth. Epi­ ample amounts of tanniniferous substances and crystals dermal cells are arched outwards in Linostoma Wall. ex of calcium oxalate. In contrast, the spongy parenchyma End!. and papillose on the lower surface in species of is usually adaxially arranged and aerenchymatous in Daphne L. most species, corresponding to the epistomatic state of the leaves. With spongy parenchyma around the stomata. Speculations on functions and ecological aspects of molecules of carbon dioxide would penetrate deep into the leaf epidermis in Passerina have been dealt with by the leaf and the large intercellular spaces of aerenchyma Bredenkamp & Van Wyk (1999, 2(00). Leaf arrange­ surrounding the vascular bundles would possibly have a ment in Passerina causes the abaxial epidermis to be moist atmosphere critical to physiological processes largely exposed to the atmosphere, resulting in epider­ such as photosynthesis, respiration and transpiration. 79

TABLE 4.-5elected morphological and anatomical characters of all leaf structural types in Passerina ~

Secon­ Main Bundle sheath Main dary Leaf vascular en veloping main Palisade ! vein vascular bundle vascular bundle bundles parenchyma Spongy Taxon Struc­ Outline Width ~m Width ~m Shape Com­ Adaxial No. No. (each Shape No. No. I parenchyma tural .. pletely cap cells side of layers cells per type ~ main bun­ 50~m « dIe)

P. sp. nov. I (Figure 3A) A transversely elliptic ± 850 ± 450 widely ovate X 19-25 3 or 4 adaxial 3 3 abaxial 4 P. burchellii BI narrowly transversely ± 1480 ± 540 widely ovate X ± 20 4 horseshoe I or 2 4 aerenchymatic elliptic p, pendll/a (Figure 3B) BI canaliculate 780--860 ± 340 widely ovate X 17-19 4-9 horseshoe I or 2 4 or 5 aerenchymatic P. comom B2 transversely elliptic ± 940 ±460 oblate X ± 22 3-5 horseshoeN 3 or 4 4 or 5 mesomorphic p, pa/udosa (Figure 3C) B2 narrowly transversely 990--1140 410--460 ovate X 19-20 3-5 horseshoe 3 or 4 aerenchymatic elliptic p , ga/pinii (Figure 2C) B2,B3 transversely elliptic 1000--1150 520--550 widely ovate X 18-25 3 horseshoe lor2 30r 4 aerenchymatic P. drakensbergensis B3 canaliculate 720--830 370--400 widely ovate X 17-19 3 or 4 horseshoe I or 2 3 or 4 aerenchymatic P. ericoides (Figure 3D) B3 transversely elliptic 910--1100 390--470 oblate X ± 22 3 horseshoe 2 or 3 3 aerenchymatic 00 0 p. sp, nov. 2 B3 depressed obovate ± 720 300--360 widely ovate X 23-28 2 or 3 horseshoe I or 2 4 or 5 mesomorphic p. sp, nov, 3 B3 canaliculate 810--880 380--430 very widely X 18-27 3 horseshoe I or 2 4 or 5 aerenchymatic ovate P. rllbm B4 canaliculale 800-920 330-400 wiuely uvale X 23-28 6 hurseshoe I ur 2 4 or 5 l1ert:m;hymali<.: P. ji./ifonnis subsp ,ji./ifonnis B3 , B4 cordiform 780-860 350--420 widely ovate X 13-15 3 or 4 horseshoe I or 2 3-5 aerenchymatic (Figure 3E) P. ji./ifonnis subsp, nov, B4, B6 transversely elliptic ± 820 ± 400 widely ovate X ± 27 2 or 3 horseshoe 2 5 aerenchymatic P. oblllsifo/ia (Figure 30) B2, B3, transversely elliptic 730--1090 370--470 widely ovate X 20--26 3 or 4 horseshoe 2 4 or 5 xeromorphic B4,B5 P. g/omerala subsp, g/omerala B2, B3 , transversely elliptic 570--980 290-500 very widely X 21 - 32 3 or 4 horseshoe 2 or 3 4 or 5 xeromorphic (Figure 3F) B5 ovate p, g/omerala subsp. nov, B6 canaliculate 770--830 420--430 ovate X 22-25 2-4 horseshoe 2 3 or 4 mesomorphic aerenchymatic p, fa/cifo/ia (Figure 4A) C carinate 900--960 520--550 obovate X 12-14 3 or 4 V lor 2 3 or 4 aerenchymatic p, sp. nov, 4 (Figure 4B) C carinate 820--1280 400--700 obovate X ± 21 3 or 4 V 2 3 or 4 mesomorphic p, monlana (Figure 4C) B2, l33, transversely elliptic 560--710 260--360 obovate X (B2, X (Dl ) ± 25 (B2, B3) 3 or 4 horseshoe I or 2 4-6 B3 ) or ± 19 (DI) 01 I:C U 1-3 5 mesomorphic 0 ,0. pa/eacea (Figure 40) B2, B3, transversely oblong, 620--860 X 400--570 obovate X (B2, X (D2) ± 23 (B2. B3, 3 or 4 50 B3. B4 ) B4) or ± 17 (02) ~ B4.02 margins erect Oi' 3-5 aerenchymatic P, rigida (Figures 3H, 4E) B3 . B6, transversely elliptic 680--780 X 270--390 obovate X (B3, X (02) 13-20 (B3, B6) 4-6 horseshoe 1-3 V> 02 (large in B6) B6) or 18-22 (02) 4 or 5 aerenchymatic N carinate 660--870 X 330--500 obovate X 14-23 3 or 4 horseshoeN 1 or 2 p, vlI/garis (Figure 4F) 02 ~ X =present, Bothal ia 3 I , I (200 J) 67

Ecological significance ber of cells and layers and specialization of tissues takes place in types B3 and B4, with B5 and B6 representing The xeromorphic character of the mesophyll can be the most xeromorphic forms. A similar increase in xero­ expressed in tenns of the number of cell layers and the morphism can be shown in structural types C and D 1 and density (reflected by the number of cells per 50 IJm) of D2, with the abaxial arrangement of the main vascular the palisade parenchyma as well as the appearance of the bundle, the increase in sclerenchyma tissue and the ulti­ spongy parenchyma (Table 4). Possible adaptation to mate formation of the sclerenchymatous hypodermal physiological drought is shown by the homogeneous and sheath, in type D2, as ·the most xeromorphic state. palisade-like mesophyll of P. sp. nov. I, which grows on the high mountains of the Karoo, where it is often cov­ ered by snow in winter. Among the remainder of the Ecological significance species, the most xeromorphic state is found in P. glom­ erata subsp. glomerata (Figure 3F), P. obtusifolia Replacement of the main vascular bundle from the ad­ (Figure 3G) and P. comosa, all growing in the NW parts to the abaxial position with the sequential increase in of Western Cape; their leaves have (1 )2 or 3 layers of xeromorphism suggests an adaptive strategy (Figure 5). cells in the palisade parenChyma, a density of 4 or 5 cells In type B the main vascular bundle is close to the adaxial per 50 ,..m and the spongy parenchyma is densely epidennis and stomata, possibly enhancing transpiration. arranged. The mesophylJ of P. ericoides (Figure 3D), It is furthennore completely surrounded by aerenchyma. P. paleacea (Figure 4D) and P. rigida (Figures 3H; 4E) Vascular tissue in close contact with aerenchyma provides can also be considered xeromorphic, possibly in water, causing a moist atmosphere and a high water response to the effect of salt spray, as these species grow potential in the large intercellular spaces. Inorganic and on the sea shore. A xeromorphic mesophyll is also indi­ organic substances are transported by the xylem and cated for P. montana (Figure 4C), which occupies a wide phloem for various pbysiological processes taking place range of habitats along the eastern escarpment. On the in the mesophyll of the leaf. The more xeromorphic other hand, P. paludosa (Figure 3C), a rare species from arrangement mechanically strengthens the leaf, allows marshy areas in the Cape Peninsula, has an inverse dor­ enough moisture for photosynthesis and respiration, but siventral leaf with one layer of palisade parenchyma, a possibly retards loss of water through transpiration. density of 3 or 4 cells per 50 IJ m and a large aerenchy­ Increasing xeromorphism is illustrated by the vascular matic spongy parenchyma., corresponding to the general bundle becoming abaxially orientated and fmally by scle­ trend towards aerenchymatic tissue in marsh plants. renchyma tissue of the vascu1ar bundle abaxially project­ ing beyond the vascular bundle sheath and reaching up to the abaxial epidermis, leaving only the adaxial part of the Leaf structural types: orientation and structure of vascular bundle in contact with aerenchyma. Vascular tis­ main vascular bundle in relation to epidermis and sue, providing moisture for the critical physiological mesopbyU processes, is thus separated from aerenchyma by one or two layers of the parenchymatous bundle sheath cap. These adaptations, associated with geographical distribu­ Prevailing characters of vascuLar bundles in tion (Table I), can clearly be illustrated at species level Thymelaeaceae and for all the leaf structural types.

In Thymelaeaceae, large portions of vascular bundles are Type A: the main and secondary vascular bundles are often occupied by sclerenchyma and surrounded by a sheath closely arranged with bundle sheaths adhering, fonning of parenchymatous cells containing tannin (Van Tieghem a central plate of veins surrounded by two or three layers 1893; Gilg 1894; Supprian ]894; Metcalfe & Chalk ]950). of palisade-like mesophyll, possibly acting as a protec­ Both these characters are present in Passerina. Intraxylary tive sheath against the minimum temperatures, which are phloem in the petiole and midrib was recorded by Leandri often below freezing point in the habitat of P. sp. nov. I (1930) and cited by Domke (1934) for many genera, (Table 1; Figure 3A). excluding Passerina (= Chymococca). The lack of intraxy­ lary phloem in leaves of Passerina was confinned by the Type B I: P. burchellii and P. pendula (Figure 3B) are present study (fables 3; 4; Figure 5). both mountainous species often surrounded by mist The main vascular bundle is more or less against the adaxial Xeromorphic gradient epidennis close to the stomata and is completely sur­ rounded by aerenchyma, possibly enhancing transpira­ The progressive change in orientation of the main tion and aeration of the leaf. vascular bundle in relation to the mesophyll shows a Types B2, B3 and B4: these are the most common leaf xeromorphic gradient (Figure 5). Leaf structural type A types, found in ± two-thirds of Passerina species (Table was defined on the basis of the isobilateral leaf, which is 4; Figure 3C-E). The possible adaptive significance of probably an ancestral state. The xeromorphic character these structural types remains much the same as in B I , of the leaf is strongly supported by the associated except that the leaf becomes sequentially more xeromor­ homogenous mesophyll and central vascular system. The phic as the main vascular bundle borders on or sinks into leaves are inverse-dorsiventral in all other structural the palisade parenchyma, possibly curtailing water loss. types. A xeromorphic gradient is clearly expressed in leaf structural type B. In types B I and B2 the mesophyll is Type B5: xeromorphism is enhanced in P. glomerata aerenchymatic and the main vasular bundle adaxially or subsp. glomerata and P. obrusijoiia (Figure 3F, G), both centrally arranged. An increase in the density, the num­ growing in the wann, arid Karoo. The main vascular

81 68 Bothalia 31 .1 (2001)

bundle is abaxially embedded in palisade parenchyma discussed the anatomical method applied by the two pre­ which differentiates into collenchyma and the spongy vious workers, doubting the constant taxonomic val ue of parenchyma surrounding the main vascular bundle 'Spicularzellen'. Thoday (1921) described a sclerenchy­ which is more densely arranged with smaller intercellu­ matous hypodermal sheath extending to the margins of lar spaces. This more xeromorphic arrangement mechan­ the leaves in P. filiformis and P. cf. falcifolia, introduc­ ically strengthens the leaf, allows moisture for pbysio­ ing the term 'wandering fibres'. Metcalfe & Chalk logical processes but possibly retards loss of water. (1950) acknowledged the previous works, also mention­ ing the presence of bundles of sclerenchymatous ele­ Type B6: found in P. glome rata subsp. nov. occurring ments supporting leaf margins in species of Daphnopsis on mountain tops from the Cederberg to the Cape C.Mart., Dicrano/epis Planch. and Passerina. Peninsula and P. filiformis subsp. nov. which grows between Malmesbwy and Vredendal. In this xeromor­ During this study variation concerning leaf structural phic leaf the main vascular bundle is adaxially surround­ types was taken into consideration and amply document­ ed by spongy parenchyma, enhancing aeration, and is ed. Considering the wide distribution of especially P. abaxially strengthened by palisade parenchyma and the montana and P. rig ida, variation in leaf structural type vascular bundle sheath which differentiates into col­ could be expected. In P. montana (Figure 4C), hypoder­ lenchyma. In P. rigida (Figure 3H), which grows in salt mal fibres are absent although lignified fibres project spray along the coast, the exceptionally large main vas­ beyond the vascular bundle sheath and reach the abaxial cular bundle is close to the adaxial epidennis and borders epidennis (type 01). Hypodermal sclerenchyma fibres on the tanniniferous abaxial epidennis, with the possible have been recorded in P. paleacea and P. rigida with leaf adaptive advantage of strengthening the leaf and allow­ structural types B2, B3 and B4 (Tables 3; 4), but without ing transpiration and associated physiological processes the development of a hypodermal sclerenchvmatous Ilt the same tilllt:. sheath. The hypodermal sclerenchymatous sheath is usu­ ally associated with leaf structural type D2 as in the fol ­ Type C: found in P. falcifolia (Figure 4A) and P. sp. lowing description. The state in which the main vascular nov. 4 (Figure 4B), growing in relatively moist environ­ bundle is abaxially orientated, the scierenchyma extending ments (Table 1). The abaxial swface of the carinate leaf beyond the vascular bundle sheath, through the inverted is in contact with the atmosphere, but the obovate main palisade parenchyma, reaching the abaxial epidermis (type vascular bundle, situated abaxially, is well protected in D) and connecting with the hypodermal sclerenchymatous the V-shaped palisade parenchyma. The adaxial fibres to form a hypodermal sclerenchymatous sheath, parenchymatous bundle sheath cap and the vascular tis­ often reaching up to the leaf margins. Leaf structural type sue are in close contact with the aerenchyma, providing D2 is regarded by the present authors as the 'ultimate' moisture for the various physiological processes. adaptation, in Passerina, to the Mediterranean climate of the Cape Floristic Region. Figures 4~F; 5. Type D 1: in P. montana (Figure 4C) the extraxylary sclerenchyma fibres touch the abaxial epidennis, but hypodermal fibres are absent. This arrangement indicates Less important taxonomic characters a high degree of mechanical strengthening and xeromor­ phism, possible adaptations to the wide range of habitats Leafwidth along the eastern escarpment where these plants grow Considering the cymbifonn, canaliculate or carinate (Table 1). shape of leaves as well as the movement of the lamina due to turgor pressure in the leaf, leaf width can at most Type D2: present in P. paleacea (Figure 4D), P. rigida be used to interpret leaf shape, but is not regarded as taxo­ (Figure 4E) and P. vulgaris (Figure 4F), all growing in nomically significant. Western Cape, the centre of diversity for PBSserina and from where certain species extend west-, north- and east­ wards. Orientation and structure of the main vascular bun­ Crystals dle ale the same as for type C, except that the sclerenchyma tissue of the vascular bundle projects beyond the vascular Calcium oxalate crystals or lime crystals were consid­ bundle sheath and reaches up to the abaxial epidermis form­ ered as taxonomically valuable in the Thymelaeaceae ing a sclerenchymatous hypodermal sheath, thus strength­ and certain species of Passerina by Supprian ( 1894). ening the leaf and making it more xeromorphic. Vascular Solereder (1908), Metcalfe & Chalk (1950) and Metcalfe tissue remains in close contact with the aerenchyma, pr0­ (1983) report the presence of both druses and crystal viding moisture for the various physiological processes. sand in the ThymeJaeaceae, but do not consider these crystals of much taxonomic value. In the present study, druses were recorded in the parenchyma cells of the mes­ Sclerenchyma ophyll in all taxa of Passerina. Calcium oxalate crystals in the intercellular spaces are regarded as fragments of Prevailing characters in Thymelaeaceae druses resulting from processing. Van TJeghem (1893) described extraxylary fibres, specially mentioning those without lignification in Tanniniferous substances Daphne mezereum L. and with lignification in D. cneo· rum L. Supprian (1894), mentioned the presence of The substances are abundantly present in the epider­ fi bres in the mesophyll of the leaves, which he called mis, mesophyll as well as vascular bundle sheath and 'Spicularzellen' and regarded as a constant taxonomic parenchyma in all taxa of Passerina. No significant character. In a subsequent paper, Gilg (1894), critically interspecific variation was recorded.

82 Bothalia 31,1 (2001) 69

Phylogenetic considerations Irregular sclerenchymatous fibres present in the meso­ phyll of leaves in many species, have been modified into Leafstructural type correlated with epidermal structure a sclereocbymatous hypodermal sheath in Passerina.

A phylogenetic gradient for the leaf structural types cannot be shown, as various characters probably evolved Genus level separately. Thoday (1921) reported that juvenile leaves in Passerina were isobilateral in transverse section, indi­ In most genera of Thymelaeaceae, leaves are isobilat­ cating the possible ancestral state of leaf structural type eral, dorsiventral or inverse-

70 BoLhalia 31, I (200 I)

CONCLUSIONS BREDENKAMP, C.L. & VAN W YK. A.E. 1999. Structure of mucilaginous epidermal cell walls in Passerina (Thymelae­ aceae). Botanical Journal of the Linnean Society 129: 22.\-2~H. Leaf anatomical evidence proved extremely useful in BREDENKAMP, CL. & VAN WYK, A.E. 2000. The epidermis in the classification of Passerina. Four leaf structural types Passerina (Thymelaeaceae): structure. function and taxonomic and ten states are associated with specific habitats and significance. Bothalia 30: 69-86. geographical distribution, illustrating a xeromorphic gra­ DOMKE, W. 1934. Untersuchungen liber die syslenraliscile IlIId geo­ dient. On the basis of leaf structural types, four new graphisehe G/iederung der Thymelaeaeeen. E. Schweizeroan'sche Verlagsbuchhandlung (Erwin Nagele) G.M.B.H., Stuugart. species and four subspecies were identified. Certain phy­ ENDLICHER, S. 1847. Generum plantarum. suppl. 4. Beck. Vienna. logenetic tendencies were proposed and the systematic ESAU. K . 1965. Plam anatomy, edn 2. Wiley. London. val ue of the various characters at family, genus and FAHN, A. 1982. Plant anatomy, edn I. Pergamon Press. Oxford. species levels were assessed, thus enabling the anatomi­ FEDER, N. & O'BRIEN, T.P. 1968. Plant microtechnique: some princi­ ples and new methods. American JOllmal ofBown.\' 55: 123-142. cal characteri zation of all infrageneric taxa in Passerina. GlLG, E. 1891. Thymelaeaceae. Die Iialiirlie/,ell Pjlanzenfamiliell 3,00: 216-245. This study illustrates prevailing Thymelaeaceae char­ GILG. E. 1894. Studien iiber die Verwandtschaftsvemiiltnisse der acters as we)) as their modification and newly evolved Thymelaeales und Uber die 'anatomische Methode'. BOlalliscile tendencies in Passerina. Observations correlate with Jahrbiicher 18: 489-574. JACKSON. B.P. & SNOWDON. D.W. 1990. Atlas of lIIicroscopv of those obtained from other studies in the genus. Based on medicinal plal1ls, cutinory herbs alld spices. Belhaven Press. the secondary reticulum of the polyforate pollen grain of London. Passerina, the subtribe Passerininae Endt. was raised to JEFFREE. C.E. 1986. The cuticle, epicuticular walles and trichomes of the tribe Passerineae (Endl.) Bredenkamp & Van Wyk plants, with reference to their structure, functions and evolu­ ti on. In B. Juniper /I' ~. Southwood. Insects alld the planl SlIr· ( 1996). The unique leaf structural types and states iden­ face: 23-M. Edwu,.: Arnold, London. tified by the present leaf anatomical study, provide more KARNOVSKY, M.J. 1965. A formaldehyde-glutaraldehyde fixative of evidence in support of the tribe Passerineae. high osmolality for use in electron microscopy. JOIITl/al of Cell Biology 27: 137a. KUGLER. H. 1928. Uber inverse-dorsiventrale Blatter. Plunta 5: 89-134. LEANDRI. J. 1930. Recherches analomiques sur Ics ThymeJeacees. ACKNOWLEDGEMENTS N males des Sciences Naturales Botanique 12: 125-237. MAUSETH, D. 1988. Plant anatomy. Benjamin/Cummings. California. We are indebted 10 Mmes H. du Plessis and C. Steyn METCALFE. C. R. 1979 . The leaf: general topography and ontogeny of and Dr E. Steyn for assistance with the LM, as well as Mrs the tissues. In CR. Metcalfe & L. Chalk, Allatomv of tile A. Romanowski for developing and printing many excel­ dicotvledol1s I, edn 2. CI3)"cndon Press, Oxford. METCALFE, C.R. 1983. Secreted mineral substances: crystals. In C.R. lent photographs. We are grateful to Prof. J. Coetzee, Mr Metcalfe & L. Chalk, Anatomy of the dicotvledol/s 2. edn 2: c.P. van der Merwe and Mr AJ. Botha, of the University 82-91. Clarendon Press, O~ord. of Pretoria, for assistance with the SEM, TEM and EDS. METCALFE. C.R. & CHALK. L. 1950. Allatomy of the dicoryledons 2, edn I. Clarendon Press, O xford. PICK, H. 1882a. Ueber den Einfluss des Lichtes auf die Gestalt und Orientirung dcr Zellen des Assimilationsgewebes. {Jot(lnische REFERENCES Zentralblatt 5.2: 400--406. PICK, H. 1882b. Ueber den Einfluss des Lichtes auf die Gestalt uno AMELUNXEN, E , MORGENROTH, K. & PICKSAK, T. 1967. Unter· Orientirung der Zellen des Assimilationsgewebcs: II. For­ suchungen an der Epidennis mit dem Stereoscan·Elektron­ demder Einnuss des Lichtes auf die angeernte Pall isaden form. enmikroskop. Zeilschrift jiir Pjlanzenplrysiologie 57: 79-95. Botanische Zentralblatt 5,2: 438-446. BARTHLOTT. w., NEINHUIS. C., C UTLER, D .. DITSCH, E, SOLEREDER, H. 1899. Systemalische allatomie der Dieorvledollell. MEUSEL, I.. THEISEN, I. & WILHELMI, H. 1998. Verlag von Ferdinand Enke. Stuttgart. Classification and terminology of plant epicuticular waxes. SOLEREDER. H. 1908. Systematic anatomy of the dicotvledoJl,l" 2. Bownical Journal ofthe Linnean Society 126: 237- 260. Clarendon Press, Oxford. BEAUMONT, A.1., EDWARDS, T.1. & VAN STADEN. J. 1994. Leaf SUPPRIAN, K. 1894. Beitriige ZIF Kenntnis der Thymelaeaceae und and involucral bract characters of systematic usc in Gnidia Penaeaceae. Botallische Ja~".biieher 18: 306--353. (T hymelaeaceae). Prcx:eedlngs of Ihe Elec/ron Microscopy THODAY, D. 1921. On the behaviour during drought of leaves of two Society of Soulh Af rica 24: 47. Cape species of Passerina, with some notes on their anatomy. BEYERS. 1.B.P. 1992. The generic delimilation of Lachnaea and Annals of Botany 35: 585-60 I. Cryptadenia (Thymelaeaceae). M.Sc. thesis. UnivelOity of Stel­ THODAY. D. 1924. XVIII. A revision of Passerina. Kt:\I" Bulletill 4: len bosch. Stellenbosch. 146--168. BEYERS. J.B. P. & VAN DER WALT, J.J.A. 1995. The generic delim­ VAN TIEGHEM, P.H. 1893. Recherches sur la structure et les affinites itation of Lac/maea and Cryptadenia (Thymelaeaceae). Botha ­ des Thymeleacees et des peneacees. Allllules des Sci('lIces lia 25: 65-85. Naturelles BOlallique 7.17: 185-294. BOND. P. & GOLDBLATT, P. 1984. Plants of the Cape flora: a de­ WEIGLIN. C. & WINTER, E. 1991. Leaf structures of "erohalophytes scriptive catalogue. Journal ofSOlllil African Botany 13: 1-455. from an east Jordalli an salt pan. Flora 185: 405-424. BREDENKAMP, c.L. & VAN WYK, A.E. 1996. Palynology of the WILKINSON, H.P. 1979. The plant surface. In CR. Metcalfe & L. genus Passerina (Thymelaeaceae): relationships. form and Chalk, Anatomv of Ihe dicoryledoll.l I. edn 2: 97-165. function. Grana 35: 335-346. Clarendon Press, Oxford.

84

4.5 Inflorescences and flowers

BREDENKAMP, c.L. & VAN WYK, A.E. 2001b. Taxonomic significance of inflorescences, floral morphology and anatomy in Passerina (Thymelaeaceae). Bothalia 31: 2] 3-236.

CONTENTS

INTRODUCTION ...... 87 MATERIALS AND METHODS ...... 90 Terminology ...... 90 Floral envelope ...... 90 Phylogeny ...... 90 RESULTS ...... 90 Inflorescences ...... 90 Bracts ...... 92 , Key to taxa based on bract characters ...... 92 Generic description of floral morphology ...... 93 Generic description of floral anatomy (as seen in transverse section) ...... 93 Floral morphology at species level ...... 97 DISCUSSION ...... 97 Inflorescences ...... '" ...... 97 Bracts ...... 97 Floral morphology and anatomy at generic level ...... 97 Receptacle ...... " ...... 97 Hypanthium and androecial position ...... 100 Identity of floral envelope ...... 101 Fragmentation ofhypanthium ...... 103 Androecium ...... 103 Separation of fused sepal and stamen traces...... 104 Pollen ...... 104 Disc ...... 104 Ovary ...... 105 Ovule type and position ...... 106

85

Obturator ...... " ...... 106 Fruit ...... 106 Seed ...... 106 Floral and fruit morphology at species level ...... 106 Fruit ...... 106 Filament length ...... 107 Anthers ...... 107 Floral envelope ...... 107 Less important taxonomic characters ...... 108 Taxonomic relationships ...... 108 Speculations on phylogeny ...... 109 Systematic value ...... '" ...... 109 Family level ...... 109 Subfamily level ...... 109 Tribal leveL ...... 109 Genus level ...... 109 Species level ...... 109 CONCLUSIONS ...... 109 ACKNOWLEDGEMENTS ...... 11 0 REFERENCES ...... 110

86 Bothalia 31,2: 213-236 (2001)

Taxonomic significance of inflorescences, floral morphology and anatomy in Passerina (Thymelaeaceae)

c.L. BREDENKAMP* and A.E. VAN WYK**

Keywords: anatomy, androecium, exotegmen. flower, gynoecium, morphology, Passerina , phylogeny, taxonomy, Thymelaeaceae, vasculation

ABSTRACT

Comparative studies were undertaken on the inflorescence, bracts and floral morphology of all taxa of the genus Pas.l'erilla L. in southern Africa. Information is given in tabular form and a key based on bract morphology is presented. Floral morphology supported the status of the infrageneric taxa and also proved to be of taxonomic significance in the genus. Controversy surrounding the interpretation of a number of floral morphological structures in Passerilla ha ~ been resolved. Morphological and anatomical evidence allowed a re·interpretation of the structure of the receptacle, hypanthium and sepals, ovary type and position, structure of the seed coat, ovule type and position, obturator, fruit and seed. On this basis an authentic generic description of the floral morphology was compiled. Passerina is distinguished by the following set of characters, a very short floral receptacle, tubular hypanthium, petaloid calyx, absence of petals and petaloid scales. diplostemonous djmorphic androecium, extrorse anthers, superior ovary, anatropous, ventrally epitropous ovule, an obturator of elongated cells, a I-seeded berry or an achene and tegmic seed with nuclear endosperm becoming cellular throughout. On this basis the flower in Passerina is considered a phylogenetically advanced structure, supporting the view that the genus is advanced within the Thymelaeoideae. The proposed taxonomic relationship between Thymelaeaceae and Malvales is confirmed by floral morphological evidence.

CONTENTS Most species of Passerina are endemic to the Cape Floristic Region and adapted to a Mediterranean or semi­ Introduction , ...... , ...... 213 Mediterranean climate. The distribution of P. sp. nov. 4 Materials and methods ...... , . , ... , . . . . . , 216 and P montana extends eastwards and northwards along Tenninology ...... 216 the eastern mountains and Great Escarpment of southern Phylogeny ...... , ...... , ...... 216 Africa, predominantly summer rainfall areas. The Results ...... ' 216 species are apparently all wind-pollinated. Inflorescences ...... 21 6 Bracts ...... 218 The controversy surrounding the interpretation of Key to taxa based on bract characters ...... 218 certain fl oral structures in the genus became obvious Generic description of fl oral morphology ...... 219 during the present study. Heinig (1951) did not incl ude Generic description of floral anatomy (as seen in Passerina in her study of the floral morphology of the transverse section) ...... 219 ThymeJaeaceae. From the sexual system of Li nnaeus (1781 , Floral morphology at species level ...... 223 1784) to Domke (1934), floral morphology played an Discussion ...... 223 integral part in the intrafamilial classification of the Inflorescence ...... 223 Thymelaeaceae and in the circumscription of the family Bracts ...... 223 (Table ] ). Between ± 1960 and 1996, vast leaps were Floral morphology and anatomy at generic level . 223 taken in the classification of the fl owering plants by Floral and fruit morphology at species level. . .. 232 making use of anatomical, floral, palynological, embryo­ Less important taxonomic characters ...... 234 logical and chemical evidence. During this period, dis­ Taxonomic relationships ...... 234 agreement on the circumscription of the Thymelaeaceae Speculations on phylogeny ...... 235 was common (Table 2). Since 1990, many higher-level Systematic value ...... , ...... 235 phylogenetic relationships in angiosperms were based on Conclusions ...... 235 evidence from rbcL and 18S nuclear ri bosomal DNA Acknowledgements ...... 236 sequence data. In many cases these studies confirmed References ...... 236 previously proposed phylogenetic patterns and trends within the family, based on morphological evidence; in INTRODUCTION other cases, however, profound changes in the circum­ scri ption and relationships of the Thymelaeaceae were The infra generic taxonomy of Passerina L. is a prob­ suggested (Table 3). lem, due to the apparent lack of marked morphological diffe rences between species. The latest revision by The present paper emanated from a monographic Thoday (1924), is now mostly outdated. study of the genus cUITen tly in progress. Available evi­ dence suggests the presence of at least four new species and four new subspecies, to be added to 16 previously recognized species, mostly endemic to southern Africa * National Botanical Institute, Private Bag X! DI, 0001 Pretoria. ** H.G.W.J. Schweickerdt Herbarium, Department of Botany, Univer­ (Thoday 1924; Bond & Goldblatt 1984) (Table 4). Here sity of Pretoria, 0002 Pretoria. we report on a comparative morphological study of the MS. received: 2000-0R-22. inflorescence, bract, flower, fruit and seed. Specific 87

..,.tv

TA BLE I ,-Classification of PasserillG within the Thymelaeaceae according to different authors, based mainly on floral, fruit and seed morphology

Rank Wikstrom 1818 Endlicher 1837, 1847 Meisner 1857 Bentham & Hooker 1880 Gilg 1891, 1894 Lcandri 1930 Domke 1934 Undefined Thymelaearum Class Thymelacae

Order Daphnoideae Thymclaeaceae Thymelaeaceae Pamily Thymelaeaceae Thyll1elaeaceae Thymelaeaceae Subfamily Thymelaeoideae Thymelaeoideae Tribe Thymelinae Gnidieae Euthymclaceae Daphneae Thymelaeoideae Gnidieae Subtribe Passerineae Diplostemoneae Passcrininae Passcriniinae Passcrininac Series Series I 00 00 Division Octandria

Inflorescence, floral, fr uit and seed morphology at gen us level

Inflorescence flowers lateral terminal spikes Floral arrangement perigynous perigynous perigynous Floral tube perigone, infundibular, calyx tube hypocrateri­ perianth al1iculate receptacle cylindric, floral lube: fusion of 4 calyx tube flask -shaped, limb 4-fid forlll, persistent above ovary 3niculate in upper half external whorls, 311iculate articulate Calyx o limb 4-pal1ite 4-lobed Corolla infundibular,4-fid, scales 0 petaloid scales 0 perianth scales 0 pctaloid scales 0 petaloid scales 0 petals 0 persistent

Androecium CA) A = 8 diplostemonous, A = 8, A = 8, exscl1cd, diplostemonous, exserted diplostemonous, A = 8 diplostemonous. A = 8 diplostcmunou s. A = 8 in incisions of perigonc alternating with sepals Gynoecium (G) unilocular, ovule unilocular, ovule single unilocular. ovule single pendulous Fruit nux nux nucula, pericarp indehiscent berry or achene crustaceous Seed I-seeded I-seeded black, crustaceous testa. til micropyle beak-like :2 ~ "'­t::' w

'tv N 8

o:l TABLE 2.-Classification of Passerinu within the Thymelucaccae according 10 different authors. based on morphological. anatomical. palynological. embryological and chemical characters 0 5­ ~ Rank Melchior 1964 Hutchinson 1973 Dahlgren 1975a. b Dahlgren 1980. Cronquist 1981. 1988 Thorne 1992a. b Takhtajan 1997 ~ . Dahlgren & Thorne 1984 ""' N Undefined Thymelaeales N Class Magnol iopsida Angiospermae 8 (Magnoliopsida) Subclass Rosidae Dicotyledonae Dilleniidae (MagnoJiidae) Superorder Thymelaeanae Malviflorae(= Dilleniiflorae) Malvanae Euphorbianae Order Thymelaeale" Thymelaeales Thymelaeales Myrtales Euphorbiales ThymeJaeales Family Thymelaeaceae Thymelaeaceae Thymelaeaceae [excluded Thymelaeaceac Thymelaeaceae Thymelaeaceae from Myrtales (1984)] Subfamily Thymelaeoideae Thymelaeoideae Thymelaeoideae Tribe Thymelaeeae (= Gnidieue) Subtribe Series Division Relationships Myrtales: perianth tube placed between Bixales placed between Dillenianae Myrtales: anatomical evi­ Myrtales: strongly Euphorbiaceae. Simmond- Gonystylaceae only other and Prote aceae (including Malvales) and dence, Euphorbiales- perigynous, polypetalous siaceae, Dichapetalaceae, family in order Malvales : pollen morphology Myrtanae Malvales: chemical. em- to apetalous flowers. Gonystylaceae bryological. palynological Other families: evidence pseudomonomerous 00 ovary, crotonoid pollen \0

Floral, rruit and seed morphology described at different ranks Character Tribe Family Order Family Family Family Flower hypugynuus, bisexual, hypogynous, bisexual perigynous,4-lllerous bisexual wind-pollinated Floral tube perianth tube articulated calyx tube hypanthium cylindrical. brightly calyx tube cylindric coloured hypanthium lobes imbricate Calyx 4-lobed perianlh tubular, 4-lobed. 4-lobed. arising frum upper imbricate rim of hypanthium petaloid appendages 0 Coro lla petals or staminodes 0 petals or scale-like pelaloid scales considered structures 0 as true petals (Dahlgren & Van Wyk 1988) diplostemonous Androecium (A) diplostemonous diplostemonous diplostemonous style sublateral; stigma (G) ovary superior unilocular. ovules pendulous, pseudomonomerous; ovules pseudomonomerous; Gynoecium pseudomonomerous papillose; carpels 2, mono­ epitropous. bitegmic, pendulous ovules solitary crassinucellate; endosperm lu~ular; ovule solitary nuclear indehiscent. nut-like, baccate, indehiscent nut or drupe indehiscent indehiscent Fruit or drupaceous, enveloped by base of persislent calyx tube caruncle-like or tail -like endosperm scanty ; embryo endosperm scanty; embryo Seed solitary appendage, without aril; straight straight embryo straighl; endo­ spem1 scanty N V> 216 Bothalia 3 1,2 (200 I)

TABLE 3.-Classification of the Thymelaeaceae according to different authors, based on molecular datu

Rank Conti el al. 1996 APG 1998* Alverson el al. 1998 Magal16n el al. 1999

Undefined Eudicots Eudicot Clade Supraordinal subgroup Core Eudicots Core Eudicots Clade (Subclass) Rosids Rosids (subclass Rosidae Rosid Clade: Geraniaceac. Rosidae) Capparales, Sapindales. Malvales, Myrtales. Core Rosids

Subgroup Expanded Malvales: Eurosids II Expanded Malvales: Core Expanded Malvales: Malvales. Thymclaeaceue, Malvales, Thymelaealean Thymelaeaceae, Sarcolaenaceae, Sarcolaenaceae, Malvales Clade, Bixalean Clade, Dipterocarpaceae. B ixaceac, Dipterocarpalean Clade Cistaccae, Sphacroccphalaceae, Neuradaceae, MlIlllillgia Order Malvales Family Thymelaeaceae (excluded Thymelaeaceae Thymelaealean clade Thymelacaceac from Myrtales) Relationships sister groups of Expanded sister group: Myrtales sister groups of Expanded sister group of Malvales: Malvales: Myrtales, Malvales: Expanded Thymelaeaceae, Sarcolacnaccae, Sapindales, Expanded Capparales, Sapindales, Dipterocarpaceae basal to Capparales Myrtales Malvales * Angiosperm Phylogeny Group.

results in leaf anatomy, indicating the arrangement of gynoecium (Heinig 1951 ; Davis 1966; Comer 1976); fru it taxa in Passerina (Bredenkamp & Van Wyk 2(01 ), are (Spjut 1994). associated with floral morphological structures as well as fru it and seed types in the present study. Floral envelope

MATERIALS AND METHODS The authors regard the floral envelope ('outer fl oral whorl') ac; a hypanthium (fused calyx and androecium), As far as possible, materiaJ was collected from at least differentiating into four petaloid sepals and a diploste­ five different localities for every taxon. Live and pre­ monous androecium, arising from the hypanthium ri m at served (dried and in liquid preservatives) material of all the separation of the sepals. For the description of colour, the species, subspecies and varieties in Passerina was texture and measurement of total floral length. only the studied (Table 4). Illustrations were made from herbari­ hypanthium and sepals are considered-the stamens are um materiaJ by means of a drawing lube. Measurements excluded. were taken by using a dissection microscope and a cali­ brated eyepiece. Phylogeny

Flowers were fixed and stored in a 0.1 M phosphate­ Speculations on phylogeny are based on prevailing buffe red solution at pH 7.4, containing 2.5% formalde­ fa mily characters representing the ancestral state and hyde, 0.1% glutaraldehyde and 0.5% caffeine [modified derived characters, indicating a reduction in tissue at Kamovsky fixative; Kamovsky (1965)]. Light micro­ genus and species levels, considered as possi bly scopy eLM) was used to study the floral anatomy of P. advanced. A cladistic analysis based on anatomical and ericoides (Bredenkamp 956, 962), which has fl eshy fru it morphological characters in Passerina is scheduled for and P. vulgaris (Bredenkamp 944, 951) with dry frui t. As the fmal stages of the study. the flowers are quite small, whole flower buds, flowers directly after anthesis and young fruits enveloped in the RESULTS fl oral tube were washed in water, dehydrated and embed­ ded in glycol methacrylate (GMA) following the meth­ Inflorescences ods of Feder & O'Brien (1968). Embedded floral mater­ ial was serially sectioned from the base of the receptacle Polytelic synflorescences present in all species; main to the anthers. Sections were stained in toluidine blue fl orescences and co-florescences spicate (Figures I A; 2A), '0'. subjected to the periodic acid-Schiff's (PAS) reaction often extended, forming multiflowered politelic fl ores­ and mounted in EnteJlan (Art. 796 1, E. Merck, Darmstadt). cences in most species, sometimes strongly reduced (P. burchellii) (Table 5). Proliferating spikes wi th inflores­ cence apex growing out and returning to vegetative growth, Terminology common; main florescences and co-florescences subtermi­ nal. Spikes sometimes artificially resembling termj nal sub­ Terminology is used according to the following capitulate inflorescences, but each characterized by two authorities: inflorescences (Weberling 1983, 1989; terminal leaves wi th axillary blind-ending rudi mentary Weberling & Herkommer 1989); hypanthium (Bunniger flowers, enveloping minute growing point (P. montana, P. 1972; Dahlgren 19753, b, 1980; Dahlgren & Thome 1984); paleacea, P. glomerata, P. sp. nov. 3); proliferating growth stamen morphology (Heinig 1951 ; Fahn 1967; Noel 1983); less common in subcapitulate inflorescences. 90

Bothalia 31.2 (2001) 217

TABLE 4.-Passerina specimens examined and housed at PRE

Species Collector Locality

bllrcllellii Thoday Bredenkamp 1545 WESTERN CAPE.-3319 (Worcester): Jonaskop, (-DC). Bollls 687*, Stokae 2542 WESTERN CAPE.-3419 (Caledon): Baviaanskloof, Genadendal, (-BA). comosa C.H.Wright Thoday 2/2 WESTERN CAPE.- 3320 (Montagu): Montagu District, near Concordia, (-CD). Andreae 1288*, WESTERN CAPE.-332I (Ladismith): Seweweekspoort, (-AD). MacDonald 2125 drakensbergensis Hillinrd Edwards 974 KWAZULU- NATAL.-2828 (Bethlehem): Royal Nlital National Park, (-DB). & B.L.Burtt Bredenkamp 1018, 1019, KWAZULU-NATAL.-2829 (Hanismith): Ndedema Gorge, Cathedral Peak Forest 1020,102 1* Reserve, (-CD). ericoides L. Bredenkamp 956· WESTERN CAPE.- 33 J8 (Cape Town): Milnerton, (-CD). Bredenkamp 962· · WESTERN CAPE.-3418 (Simonstown): Cape Maclear, (- AD). Taylor 4042 WESTERN CAPE.-3419 (Caledon): Pearly Beach, (-CB). falcifo/ia C.H.wright Brt!denkamp 9I 7* WESTERN CAPE.-3323 (WiUowmore): Gouna State Forest, (-CC). Bredenkamp 915 WESTERN CAPE.-3324 (Steytlerville): opposite Tsitsikama Lodge, (-CD). Tyson 1449 WESTERN CAPE.-3423 (Knysna): Knysna, (-AA). filifonl/is L. subsp.filifonnis Boucher 2833 WESTERN CAPE.- 3118 (Van Rhynsdorp): Clanwilliam Di s!., Diepkloof S of Verlorc­ vlei, (-AD). Bredenkamp 1039* WESTERN CAPE.-3318 (Cape Town): Signal Hi ll , (-CD). filiformis L. subsp. nov. Schlechter 5125" WESTERN CAPE.-3218 (Clanwilliam): Alexander's Hoek, (-BC). Taylor 1542 WESTERN CAPE.-32 18 (Clanwilliam): Malmesbury Div., Vredenburg, Steenberg's Cave, (-CC). ga/pinii C.H.Wright Bredenkamp 946* WESTERN CAPE.-3420 (Bred.asdorp): De Hoop Nature Reserve, (-AD). Bredenkamp 932 WESTERN CAPE.-342I (Riversdale): Riethuiskraal, (-AD). Bredenkalllp 933 WESTERN CAPE.-342 I (Riversdale): Still Bay, (-AD). Bredenkamp 923 WESTERN CAPE.-3422 (Mossel Bay): Mossel Bay, (-AA). glomerala Thunb. subsp. Bredenkamp 988 WESTERN CAPE.-3219 (Wuppertal): Cederberg Mountains, Eikeboom, (-AC). glomerata Bredenkamp 994 WESTERN CAPE.-32I 9 (Wuppertal): Cederberg Mountains, Kromrivier, (-AC). Bredenkamp 1002 WESTERN CAPE.-3219 (Wuppertal): Cederberg Mountains, Algeria, (-AC). Bredenkamp 984, 985* WESTERN CAPE.-3219 (Wuppertal): Citrusdal, PiekenierskloofPass, (-CD). Bredenkamp 977 WESTERN CAPE.-3219 (Wuppertal): Groenfontein, (-DC). Bredenkomp 973" WESTERN CAPE.-3319 (Worcester) : Thlbagh, (-AC). glomera/({ Thunb. subsp. nov.Stokoe 8040 WESTERN CAPE.-3219 (Wuppcrtal): Cederberg Mountains. Esterhuysen 28587* WESTERN CAPE.-3319 (Worcester): Hex River Mountains. 1/I0llla110 Thoday Giess 13136 NAMIBIA. -2217 (Windhoek): Auas Mountains, Molteblick, (-CA). Bredenkamp 1024 MPUMALANGA.- 2430 (Pilgrim's Rest): World's View, (-DO). Bredellkamp 1025 MPUMALANGA.- 2430 (Pilgrim's Rest): God's Window, (-DO). Bredenkamp 889, 890 FREE STATE.-2828 (Bethlehem): Golden Gate National Park, (-DA). Bredenkamp 893" FREE STATE.-2927 (Maseru): Ladybrand, (-AB). OhlusifoJia Thoday Bredenkamp 971 WESTERN CAPE.-3319 (Worcester): Karoo National Botanical Garden, (-CB). Bredenkamp 967 WESTERN CAPE.-33 19 (Worcester): Jonaskop, (-CD). Bredenkamp /033, 1034 WESTERN CAPE.-332I (Laingsburg): Seweweekspoort, (-AD). Bredenkamp 929 WESTERN CAPE.-3321 (Ladismith): Rooiberg, (-CB). Bredenkamp 919' WESTERN CAPE.-3322 (Oudtshoom): Perdepoon, (-CD). palcaceo Wikstr. Bredenkamp 960" WESTERN CAPE.-34I 8 (Simonstown): Kommetjie, (-AB). Bredenkamp 961 WESTERN CAPE.-3418 (Simonstown): Cape Maclear, (-AD). BretlellA '!IIp 952 WESTERN CAPE.-3418 (Simonstown): Harold Porter National Botanical Garden. (-BD). Bredenkamp 950 WESTERN CAPE.-3420 (Bredasdorp): De Hoop Nature Reserve, (-AD). Bredenkomp 949 WESTERN CAPE.- 3420 (Bredasdorp): Waenhuiskrans, (-CA). Bredenkamp 940 WESTERN CAPE.-342I (Riversdale): Puntjie, (-AC). pailldosa Thoday Bredenkalllp 1035 WESTERN CAPE.-3418 (Simonstown): Rondevlei ature Reserve, (-BA). Jangle 156* WESTERN CAPE.-)4l8 (Simonstown): Rondevlei Nature Reserve. (-BA). Thoday 100 WESTERN CAPE.-3418 (Simonstown): Riet Valley, Cape Flats, (- BA). pel/dl/ill Eckl. & Zeyh. Faurcade 3043 EASTERN CAPE.-3324 (Steytlerville): Zuur Anys, (-CB). Bredenkamp 908, 909* EASTERN CAPE.-332S (Port Elizabeth): Groendal Nature Reserve. (-CB). rigida Wikstr. Wa rd 72 I I KWAZULU-NATAL.-2832 (Mtubatuba): St Lucia Park, (-AD). Bredenkamp 1013* KWAZULU-NATAL.-3I30 (Port Edward): Umtamvuna River Mouth, (-AAl. Bredenkamp 899 EASTERN CAPE.-3326 (Grahamstown): Kenton-on-Sea, (-DA). Bredenkomp 898 EASTERN CAPE.-3326 (Grahamstown): Port Alfred, (-DB). BredeT/kamp 897 EASTERN CAPE.-3327 (Peddie): Kleinmond West, (-CA). Bredellkamp 91 I EASTERN CAPE.-3424 (Humansdorp): Jeffreys Bay, (-BB ). mbru C.H.Wright Bredenkomp 914" EASTERN CAPE.-3324 (Steytlerville): en route to Kareedouw, (-CD). Bretlenkamp 905 EASTERN CAPE.-3325 (Port Elizabeth): Colchester, (-DB). Bredenkamp 900 EASTERN CAPE.-3326 (Grahamstown): Grahamstown, (-AD). rulgaris Thoday Bredenkamp 926 WESTERN CAPE.-332I (Ladismith): en route to Korenthe River Dam, (-CC). Bredenkamp 907 EASTERN CAPE.-3325 (Port Elizabeth): Groendal Nature Reserve, (-CB). Bredenkamp 901 EASTERN CAPE.-3326 (Grahamstown): Grahamstown, (-AD). Bredenkamp 95/· WESTERN CAPE.-3418 (Simonstown): Betty's Bay, Harold Porter NBG, (-BD). Bredenkamp 944·* WESTERN CAPE.-3420 (Bredasdorp): Cape Infanta, (- BD). Bredellkamp 924 WESTERN CAPE.-3422 (Mosselbaai): Kleinbrak, (- AA). sp. nov. I Goldblall & Manning 8627 WESTERN CAPE.-3220 (Sutherland): RoggeveJd Escarpment, (-AB). Bredenkamp 1044,1046, WESTERN CAPE.-33 19 (Worcester): Waboomberg, Ceres, (-AD). J047 Oliver 9281* sp. nov. 2 Esterhuysen 12189, 26859* WESTERN CAPE.-32 18 (Clanwilliam): Northern Cederberg Mountains, (-BB ). sp. nov. 3 Stokoe 9302 WESTERN CAPE.-3322 (Oudtshoom): Swartberg Pass, Prince Albert area. (-AC). Schleciller 5846 WESTERN CAPE.-3322 (Oudtshoom): Montagu Pass, (-CD). &Ierhllysen 10734* EASTERN CAPE.-3323 (Willowmore): Kouga Mountains. (-DA). Esterhuysen 28006* EASTERN CAPE.-3324 (Steytlerville): Cockscomb, Uitenhage area, (-BD). sp. nov. 4 Killick 238 KWAZULU-NATAL.- 2930 (Pietennaritzburg): Table Mountai n, (-CB). Bredenkamp 1016, 1017 KWAZULL'-NATAL.-3030 (Port Shepstone): Oribi Gorge, (-CB). Vo n Wyk & Bredenkamp KWAZULt:-NATAL.-3130 (Port Edward): Umtamvuna River Bridge, (-AA). I, 1012 Bredel/kamp 1327* KWAZULlJ-NATAL.-3130 (Port Edward): Mkambati Nature Reserve, (-AC). Gillell 4537 WESTERN CAPE.-3422 (Mossel Bay): en route to Knysna from George, (-BB). Keel s.n. WESTERN CAPE.-3423 (Knysoa): Knysna, Redlands, (-AA). " Illustrated specimens; • spec imens used for light microscopy. 91 218 Bothalia 31.2 (2001)

FIGURE 1.-Passeril1a ericoides. A-G, Bredenkamp 962. A, young inflorescence; B, leaf exposing adaxial surface; C, bract exposing adaxial sur­ face; D, fruiting branchlet; E, flower enveloped by bract; F, fleshy one-seeded berry; G, seed with black tegmen and white fragment of endocarp. A, D, x 10; B, C, E-G, x 20. Scale bars: 10 mm.

Bracts mm; bracts with leaf-like point, length x liz width (4.0-) 5.1-6.3(-7.3) x (1.4-)1.5-2.0(-2.6) mm; outside glabrous, Arrangement subterminal in proliferating spikes, seldom tomentose (P. comosa, P. sp. nov. 3), inside, base pseudoterminal in subcapitulale spikes; youngest bracts or midrib tomentose with glabrous wings or completely closest to growing point, enlarging along florescence comose, tomentose, villous or setose; trichomes nong­ axis; bracts enveloping flowers and frui ts (Figures I A, landular, uniseriate, mostly strongly spiralled, white; D, E; 2A, D), largest after anthesis of flowers, becom­ coriaceous or chartaceous, rugose or smooth, ± succu­ ing more coriaceous and rounded at frui t set, gradually lent or thin, outline of epidermal cells often macroscop­ acquiring leaf shape at proximal end of florescence ically visible ; often ± 3-5-ribbed or reticuJately veined axis.. decussate, sessile, imbricate, often conspicuously on each side of main vein. Wings absent, or bordering enlarged, resulting in longer spikes (P.falcifolia, P.fili­ lamina or distinct from lamina and conspicuously formis, P. sp. nov. 4, P. rubra). Lamina inversely ericoid; rounded or bullate; mostly glabrous, coriaceous, charta­ adaxial surface (inside) concave, facing inflorescence ceous or membranous, often straw-coloured and rosy­ axi s, abaxial surface (outside) convex; cymbiform (boat­ tinged. Base sessile or cuneate. Midrib strongly devel­ shaped) or helmet-shaped; in outline (plane shape), ob­ oped, forming spine of cymbiform bract, often keeled, long, lanceolate, ovate and obovate to widely ovate and extending to form a leaf-like point in many species. obovate, rhombic and narrowly obtrullate to obtruUate Apex obtuse, rounded or acute, mostly coriaceous. (Figures 3; 4; Table 5); bracts without leaf-like point, Margins often ciliate to setose (P. burchellii, P. sp. nov. length x 1/2 width (2.5-)3.5-4.5(-5.5) x (0.9-)1.0-1.5(-2.4) 4, P. pendula).

T FIGURE 2.-Passeril/u vlligaris. Bredenkw/lp 944. A. inflores­ I /JIi,; ·. ce nce: B, leaf exposing aoax­ inl surface: C. bract exposing abaxial surface: D, flower enveloped by bract; E, fruit enveloped by hypanthium. with fragmental ion at neck 1[(~ll base; F, achene: G. seed with black tegmen and while ',! " I spots. A , x 10; B-G, x 20. A·~ c Scale bars: 10 mm.

Key to taxa based on bract characters (Figures 3; 4; Table 5)

I a Bracts hairy outside ...... P. comosa (Figure 3A), P. sp. nov. 3 (Figure 3B, C) I b Bracts glabrous outside: 2a Bracts shorter than 4.5 mm : 3n Bracts rhombic in outline: 4a Bracts dark green when fre sh, dark brown in dried specimens, coriaceous; membranous wings absent...... P. burchellii (Figure 3D) 4b Bracts blue-green (glaucous) in fresh and dried specimens, softly coriaceous; membranous wings present ...... P. pelldula (Figure 3E) 92 Bothalia 31.2 (200 I) 219

3b Bracts variously shaped, but not rhombic in outline: 5a Membranous wings present: 6a Wings present as membranous rims along lamina margins: 7a Lamina convex, ovate to obovate, thinly coriaceous, obscurely ribbed...... P. mOl1lalla (Figure 3F) 7b Lamina helmet-shaped, widely obovate, thinly chartaceous, smooth ...... P. sp. nov. 2 (Figure 3G) 6b Wings va riously shaped, but not as membranous rims along lamina margins: 8a Wings widely ovate, evenly concave. main vein forming small, obtuse apex. .. . P. paleacea (Figure 3H) 8b Wings oblate or obovate. often bullate: 9a Main vein extended into leaf-like point ...... P. ga/pinii (Figure 31) 9b Main vein shortly extended into subacute apex .. . P. sp. nov. I (Figure 3J) 5b Membranous wings absent: lOa Bracts oblong in outline, apex obtuse .. ... P. ericoides (Figure 3K) lOb Bracts widely ovate or obovate to widely obovate in outline, apex various: Ila Bracts widely ovate with distinct, short acute point. glaucous. coriaceous; lamina with 2 or 3 shallow folds ...... P. rigida (Figure 3L) II b Bracts obovate to widely obovate, point obtuse to acute, thickly coriaceous; lamina ribbed and reticu­ lately veined ...... P. g/omerata subsp. glome rata (Figure 4A), P. glomerata subsp. nov. I (Figure 4B) 2b Bracts longer than 4.5 mm: 12a Bracts lanceolate and glaucous...... P. drakensbergellsi.f (Figure 4C) 12b Bracts variously shaped and coloured, but not lanceolate and glaucous: 13a Main vein shortly extended into an acute apex; lamina rhombic 10 obtrullate. distinctly angled, 4- or 5­ ribbed...... P. vulgaris (Figure 4L) 13b Main vein extending beyond lamina into a leaf-like point: 14a Leaf-like point obtuse at apex; lamina closely 2-ribbed at margins...... P. obtusiJolia (Figure 4D) 14b Leaf-like point not obtuse; lamina extending into a membranous margin: 15a Adaxial (inner) surface of bracts basally to centrdlly setose or tomentose over entire length of midrib; wings glabrous: 16a Midrib and leaf-like point stout and strongly de veloped, apex acute ...... P. paludosa (Figure 4E) 16b Midrib shortly extended or forming a straight or filiform. leaf-like point: I7n Midrib shortly extended, wings ovate·acuminate, gradually narrowing to a point ...... P.filiJonnis subsp. nov. (Figure 4F) 17b Midrib extended. fonning a straight or filiform, leaf-like point: lSa M idrib extended into a filiform. slightly falcate point; wings widely obovate, narrowing abruptly into a leaf-like point. P.filifomlis subsp.filiJormis (Figure 4G) 18b Midrib extended into a straight point or slightly incurved point; wings ovate with margins hairy in upper half. or obtrullate. narrowing abruptly into a leaf-like point .... P. sp. nov. 4 (Figure 4H, I) ISh Adaxial (inner) surface of bracts completely villous: 19a Midrib extended, leaf-like point falcate; wings ± 4-ribbed ...... P. falcifolia (Figure 4J) 19b Midrib shortly extended in to a short point, apex acute; wings ± 5-ribbcd . . . P. nlbra (Figure 4K)

Generic description of floral morphology sepaJous whorl (1.2-)1.4-2.2(-2.4) mm long; anthers (0.5-)0.7-0.9(-1.1) x (0.2-)0.3-0.4(-0.7) mm, subbasi­ Flowers actinomorphic, bisexual, hypogynous. Floral fixed, 2-thecous and 4-locular (Figure 9A), extrorse. envelope membranous during pollination and yellowish Disc absent. Ovary superior, (l.6-)2.0-2.5(-2.7) x in P. rigida, P. paleacea, P. sp. nov. I and P. sp. nov. 2, (0.5-)0.6-1.4(-1.7) mm, bicarpellate during embryonic slighUy succulent and greenish in P. ericoides, mostly stage (Bunniger 1972), pseudomonomerous (Heinig yellow-pink in all other species, dehydrated after shed­ 1951) at maturity, placentation parietal, uniloculate, with ding of pollen, becoming papyraceous or coriaceous, I pendulous ovule laterall y attached near top of ovary; yellow-pink tones turning red , (4.0-)5.3-7.3(-8.4) mm style separating laterally from top of ovary, maintaining long. Pedicel very short or absent. Receptacle very short. lateral position in hypanthium neck. reaching beyond Hypanthium a membranous to coriaceous cylindric tube; hypanthium rim; stigma ± globose, mop-like or pen icil­ indumentum variable in density, trichomes nonglandular, late (wind-pollination). Fruit enveloped by persisten t, uniseriate, often spiralled, whitish, density of indumen­ loosely arranged hypanthium fragmented at neck base or, tu m at ovary ranging from glabrous to tomentose or in some species, fragmenting over widest circumfere nce strigose; neck (narrowed tube between apex of ovary and of fruit, shedding fragmented hypanthium, sepals and sepals) (0.3-)0.6-2.6(-3.0) mm long, density of ind u­ androecium (Figures J D ; 2E), in P. ericoides (Figure 1F) mentum ranging from glabrous to tomentose on outside, and P. rigida a fleshy I-seeded berry, 5.3 x 4.0 mm, in inside often hairy, abscission tissue not macroscopically all other species (Figure 2F; Tables 6, 7) an achene, peri­ discernible, articulation plane absent, after fruiting frag­ carp membranous and dry, 2.5 x 1.2 mm. Seed: tegmen mentation of neck base caused by dehydration and torsi­ black and shiny, often with white spots, broadly fusiform fi cation of tissue, shedding sepals and androecium wi th outgrowths at both micropylar and funicul ar ends (Figure 2D, E; Table 8) in most species. Sepals petaloid (Figures lG; 2G), 2.2(-2.9) x 1.2(-1.6) mm. (Figures IE; 2A, D); lobes 4, imbricate in bud (Figure 8e , D), flexed in flower, often setose with up to 5 long Generic description of floral anatomy (as seen in trichomes on outer surface and glabrous to tomentose on transverse section) inner surface; outer lobes cymbifonn or concave; inner lobes oblong, ell iptic or obovate. Corolla absent. Receptacle base: vascular tissue arranged in a central Petaloid scales absent. Androecium di morphic dip]oste­ stele from which 8 traces are derived in a single whorl monous, inserted at rim of hypanthium, filaments of (Figure SA). Receptacle at apical position: carpellary antipelalous whorl (0.4-)0.7-1.2(-1.5) mm long, anti­ bundles arranged in continuous central cylinder; 8 vas­ 93 Bothalia 31,2 (2001) 220

A

D

~ rl ---r---r--+-~

G

J L

FIGURE 3.-----{:amera lucida drawings of abaxial, lateral and adaxial views of bracl~ of Passerina taxa, arranged according to ~equence in key. A. P. CO/llosa, Alldreae 1288; B-C, P. sp. nov. 3, ESlerituysen 10734, 28006; D, P. burchellii, Bolus 687; E, P. pendl/la, Bredellkaml' 908; F. P. montana, Bredenkamp 893; G, P. sp. nov. 2, ESlerhuysen 26859; H, P. paleacea. Bredenkamp 960; I, P. gulpillii, Bredenkamp 946; J, P. sp. nov. I. Oliver 9281 ; K, P. ericoides, Bredenkamp 962; L, P. rigida. Bredenka1/1pIO/3. A-L x 20. Scale bar: 5 mm . cular bundles, fused sepal and stamen traces occupying each sepal containing 3 vascular bundles (Figure SA, peripheral posi tion (Figure 5B); cells at perifery of cor­ B); each of the 4 fu sed sepal midrib and antisepalous lex arranged in rows (abscission tissue), differentiation stamen bundles (s-sst) split into a sepal midrib bundle (s) of inner epidennis of hypanthium and outer epidermis of and an antisepalous stamen bundle (sst), forming the ovary wall, separating hypanthium from ovary wall second whorl of 4 antisepalous stamens (Figure 8B, C); (Figure 5C, D). Hypanthium irregularly lobed or scal­ anthers extrorse, with wall of locule comprising epider­ loped; outer and inner epidennis variously hairy; cuticle mis and endothecium only, pericJinal wal ls of epidermis sometimes strongly developed; 8 vascular bundles thin and fo lded inwards, cell wall thickenings of stretching over entire length (Figure 5E, F). Calyx with endothecium ± stellate, with rib-like extensions directed 4 imbricate lobes developing at hypanthium rim, each towards epidermis (Figure 9B), partitions between containing 3 vascular bu ndles; epidennal and hypoder­ loculi withered and ru ptured (Figure 9C), accompanied mal layers containing large amounts of pigment; spongy by final rupturing of outer walls of thecae (Figure 9D). parenchyma aerenchymatic (Figure 8C, D; Table 6). Ovary base: wall independent of hypanthium or loosely Androecium: each of the 4 fused commisural sepal and adhering to hypanthium a1 distal side away from placen­ antipetalous stamen bundles (cs-pst) split into 2 sepal ta (Figure 5E, F; Table 6); outer and inner epidermal lateral bundles (sl) and I antipetalous stamen bundle walls strongly developed, containing ample amounts of (pst), resulting in the first whorl of 4 antipetalous sta­ tanniniferous substances (Figure 5E) or less sturdy mens (situated slightly lower in the hypanthium) and (Figure 5G); mesophyll of densely arranged parenchy­ 94 Bothulia 31.2 (200 I) 221

FIGURE 4.- Camera lucida drawings of abaxial, lateral and adaxial views of bracts of Passerina taxa, arranged according to sequence in key. A. P. glomerl!ta subsp. glomerata , Bredellka1l1p 973; E, P. glomerata subsp. nov., Esterhuyscl1 28587; C, P. drakensbergellsis. Bredellkamp 11112 ; D, P. o/Jtusifo/ia, Bredenkamp 919; E, P. paludosa, Jangle 156; F. P. filiformis subsp. nov., Schlechter 5125; G, P.jiliJol1l1is subsp. jilifonllis, Bmlellkamp 1039; H. P. sp. nov. 4 from Knysna, Gillell 4537; I, P. sp. nov. 4, Bredenk(l1l1jJ 1327; J, P..falci{o/ia. Bredenklll1ljJ 917; K, P. rubra, Bredenkam{J 914; L, P. vulgari." Bredenka1l1{J 944. A-L. x 20. Scale bar: 5 mm.

95

IV TABLE 5.-Compari son of inflorescences and bracts in Passer;lIa . Sequence of taxa according lO key based on bracl chara cters (see text) IV IV

Taxa Polytelic inflorescence Cymbiform bracts Spikes Multi­ Tm,","m Size Plane shape Lamina Wings subcapitulale I reduced flowered abaxial adaxial length x 1/2 width (mm) (per side of main (per side of main vein) main and co­ vein) t10rescences

comosa X tomcntosc comosc (3.0-)5.5 x 1.5(-1.8) widely ovate. apex acute ± 3-ribbed. margins submembranous coriaceous, rugose sp. nov. 3 X tomentose comose (4.5-)4.9 x 1.5(-1.8) widely ovate, apex ac ute ± 3-ribbed, margins submembranous coriaceous. rugose

burellellii X villous (3.2-)3.5 x 1.5 rhombic. apex involute. acute coriaceous, smooth smooth pendula X comose (30-)4.2 x 1.0(-1.5) rhombic, apex obtusely softly coriaceous, margins membrdnous angled smooth mOIl/alla X villous (3. 2-)4.0 x 0.9(-1.6) ovate to obovate, apex thinly coriaceous, margins membranous subacute obscurely ribbed paleacea X villous, wing (2.5-)2.7 x 1.2(-19) widely ovate, apex obtuse chartaceous, smooth membranous, obscurely borders glabrous veined galpillii X midrib tomentose. (3.6-)45 x I. I (-2.2) oblate, leaf-like point, chartaceous membranous, broadly wings glabrous apex subacute rounded. bullate sp. nov. X midrib tomentose, (3.2-)3.5 x 1.4(-1.9) widely obovate, apex chartaceous membranous, bullate wings glabrous subacute smooth. membranous rims '-0 sp. nov. 2 X setose 3.lxl.4 helmet-shaped, widely thinly chartaccous. 0\ obovate. apex subacute smooth er;coides X villous 3.6 x 1.5 leaf-like, oblong, obtuse smooth, slightly absent succulent rigida X villous (2.6-)3.4 x 1.4(-1.9) widely ovate, distinct, coriaceous, 2 or absent short, acute poi nt 3 shallow folds glomera/a subsp. glomera/a X villous 4.0 x 1.4(-2.4) obovate to widely thickly coriaceous, absent obovate, point obtuse ribbed and reticu­ lutely vie ned glomera/a subsp. nov. X vil lous (25-)2.9 x 1.5 obovate to widely thickly coriaceous, absent obovate, point obtuse to acute ribbed absent drakensbergemis X villous 5.5 x 1.5 lanceolate, apex obtuse to coriaceous, obsurely acute ribbed and reticu­ lately veined coriaceous, closely absent ob/lls!{olia X setose (4.0-)5.8 x 1.5(-1.7) narrowly obtrullate, point Icaf-like. apcx obtllse 2-ribbed at margins coriaceous. ± 2-ribbed membranous with obscure pailldosa X basally setose. (5.2-)7.0 x 1.8(-2.0) narrowly obtrullate, wings glabrous point leaf-like, apex acute venation coriaceous membranous with distinct /iliformis subsp. nov. X centrally setose. 4.6 x 1.5 ovate acuminate, wings glabrous gradually narrowing to poi nt venati on chartaceous membranous with distinct filiforlllis subsp. filifonnis X basally to centrally 7.3 x 2.0 widely obovate, setose. wings narrowing abruptly in to venation glabrous filifonn point basally setose. 6.3 x 1.6 obtrullate, narrowing coriaceous chartaceous, ± 3-ribbed, sp. nov. 4 X obscurely veined wings glabrous abruptly into straight. co leaf-like point 0 widely ovate, narrowing coriaceous coriaceous. ± 4-ribbed. :;. falcifolia X villous (5.3-)5.8 x 1.7(-2.0) ::. into falcate point reticulately veined t;' coriaceous. ± S-ribbed. villous (4.3-)5.1 x 1.8(-2.0) widely ovate. with coriaceous Vol mbra X reticulately veined short. acute point i0 coriaceous. distinctly 4- or X shortly villous (44-)5.1 x 1.4(-1.7) rhombic to obtrullate. coriaceous N vulgaris S-ribbed 0 distinctly angled 0 X, character present.

BothaJiu 31,2 (2001) 223

TABLE 6.-Distinction between P ericoides and P vulgaris based on floral anatomy, seen in tis (Figures 5; 7: 10)

Floral part P ericoide,· (fruit fleshy) P vulgaris (fruit dry)

Hypanthium Lobed. Scalloped. Outer and Inner epidermis hairy (Figure SE-G). Outer epidennis hairy, inner epidennis glabrous (Figure SF, H).

Ovury wall Independent of hypamhium. Loosely adhering to hypanthium di stally away from placenta (Figure SF). Outer and inner epidennal wall strongly developed, Epidennal walls less sturdy with less tanniniferous substances. containing tanniniferous substances. Mcsophyll of densely arranged parenchyma cells. Mesophyll aerenchymatic. Dorsal carpellary bundle strongly developed (Figure SG). Dorsal carpellary bundle rudimentary or absent (Figure SF, H). Commissural bundles densely arranged , often fused Commissural bundles closely arranged, often separate (Figure SE, G) (Figure SF, H).

Style Four-lobed. Elliptic. Vascular bundles 4, 1 per lobe (Figure 7C). Vascular bundles inconspicuously arranged in a row along long axis of elliptic style (Figure 7D).

Fruit Pericarp fle shy: exocarp tanniniferous, mesocarp of a few Pericarp membranous: exocarp degenerated, mesocarp lacking, layers of parenchyma, endocarp disi ntegrating with outer endocarp degenerated (Figure 10D). integument (Figure IDA, B).

lila ct!lI s (Figure 5E, G) or aerenchymatic (Figure 5F, Floral morphology at species level H); vascular bundles arranged in a single ring-like whorl (Figure 5B), median and dorsal carpellary bundles sep­ Floral morphological characters and taxonomically arating from stele (Figure 5C, D), vascular bundles dif­ important fruit characters are summarized in Table 7, and ferentiating into dorsal carpellary bundle as well as specialized hypanthium and sepal characters in Table 8. median and commissural carpeJlary bundles (Figure 5E, All these are associated with specific leaf anatomical characters (Bredenkamp & Van Wyk 200 I). Q), or dorsal carpellary bundle absent or poorly defined (Figure SF, H); funiculus ventral (Figures 5; 6); ovule bitegmic, outer integument (oi) consisting of outer epi­ DISCUSSION dennis (oeoi), mesophyll and inner epidermis (ieoi), inner integument consisting of paJisade-like outer epi­ Inflorescences dermis (oeii), mesophyll and tanniniferous inner epider­ mis (ieii) (Figures 5G, H; 6A. B), crassinucellate; Weberling (1989) regards polytelic synflorescences as embryo sac with 1 cell of functional macrospore (Figure dominant within the Thymelaeaceae. He found monotelic 5G, H). Ovary al median posilion: ovule wall, integu­ synflorescences in the Gonystyloideae, a relatively primi­ ments and nucellus remaining the same; embryo sac tive group, as well as certain genera of the Thymelaeoideae with 2 cells of functional macrospore (Figure 6A, B). and Aquilarioideae. In the Gnidieae. it was found in Ovary al apical position: placental vascular bundles and Lachnaea L. (= Cryptadenia Mei sn.) (Beyers & Van der funiculus strongly developed; ovu le anatropous, posi­ Wal t 1995; Beyers 1997), a genus endemic to the Cape tion ventrally epitropous; obturator of elongate ceils Floristic Region (Beyers 1992). Weberli ng (1 989) never­ extending from base of style to micropyle (Figures 6C, theless concluded that it appears impossible to draw any D; 7 A, B); outer integument horseshoe-shaped, opening taxonomic conclusions from the existence of monotelic close to funiculus facing placenta (Figures 6C; 7 A, B); synflorescences within these taxa_Passerina is character­ micropyle formed by inner integument, initially facing ized by po)ytelic synflorescences_ Most species have multi­ upwards (Figures 6C, D; 7 A), but close to base of style, flowered main and co-florescences, and a reduction of incurv ed towards placenta (Figure 7B). Style four-lobed florescences to single and subcapitulate spikes is clearly or elliptic, stylar channel well developed (Figure 7C, D). shown (Table 5). lined by conducti ng tissue; vascular bundles 4, I per lobe (Figure 7C) or inconspicuously arranged in a row Bracts along the long axis of elJiptic style (Figure 7D; Table 6). In their descriptions of the Thymelaeaceae, Domke Sligma reaching beyond hypanthium ri m, penicillate. (1934) reports the presence or absence of bracts and bracte­ ramified into nu merous simple papillae, dispersed oles, sometimes involucral, and Peterson (1978) mentions between filament bases (Figure 8A-C)_Seed exotegmic, that deciduous or persistent bracts are often present. In outer integument undergoing atrophy; outer epidermis Passerina. single flowers are always enveloped by persis­ of inner imegument (oeii) lengthening and becoming tent bracts. In the present study, this constant taxonomic palisade-li ke, mesophy1\ consisting of 2 or 3 layers of character has been employed in a key for application in parenchyma, inner epidermis of inner integumen t (ieii) herbarium and field work (Figures 3, 4; Table 5). tanniniferous (Figure lOA, B); tegmen black. lignified. sti ll portraying palisade origin (Figure tOC, D); outer Floral morpbology and anatomy at generic level layer of nucell us ornate with cellulose thickenings. nucell us 3-5 cell layers thick; endosperm formation Receptacle nuclear (Davis 1966), but later becoming cellular throughout (Figure IOD) , absorbed by cotyledons con­ Historically the interpretation of the receptacle in the taining no starch but copious amounts of oil. Thymelaeaceae has been controversial. Tables I and 2

97

, •.> IV....

TABLE 7.-Floral morphological and fruit characters in Passeril1a. correlatcd with leaf anatomy

Fruit Horal envelope (hypanthium and sepals) Ovary Filaments Anthers *Leaf structural Fleshy Ory Colour at pollination Texture Total Length Anti- Anti- Length Taxon scpalous x width type green yellow Iyellow-pink coriaccousI mcm­ I papy­ length x width petalous I branous raceous in mm in mm 10 mm in mm inmm sp. nov. I A X X X X 5.3 L7 x 0.5 OA 1.2 0.5 x 0.3 ericoides 63 X X X 5.0 2.1 x 1.7 0.5 i.3 0.9xO.7 rig ida B3. B6. 02 X X X 4.0 2.2 x I A 0.5 1.5 0.8 x OA paleocea 62. B3 , B4. 02 X X X 4.2 2A x IA 0.7 IA 0.6 x 0.5 sp. nov. 2 B3 X X X 4.6 1.8 x 0.5 OA 1.2 0.7 x 0.3 galpillii B2.B3 X X X 5.3 2.0 x 1.1 IA 2.1 0.6 x 0.5 X 6,4 2,4 x 0.9 0.9 IA 0.8 x 0,4 \0 glomera/a subsp. glomera/a B2.B3. B5 X X 00 glomerara subsp. nov. 6 6 X X X 4.9 2.5 x 0.9 1.2 I A 0.9 x OA ol)/Llsifolia 62.63, B4,B5 X X X 6.8 2.5 x 1.1 1.2 2.2 1.1 x 0.5 burche"ii 61 X X X 4.7 1.6 x 0.6 0.7 1.5 0.5 x 0.3 pel/du/a B2 X X X 6.5 2.0 x 0.7 1.5 2.0 0.8 x OA COlll osa B2, 63. 01 X X X 7.3 2.7 x 1.0 0.7 1.5 0.7 x OA pallldo.HI B2 X X X 7.2 2.3 x 1.0 1.2 2A 0.7 x 0.5 mOl1/(1110 62. 63.01 X X X 5.8 2. 1 x 0.6 0.6 1.5 0.8 x 0.4 0.7 x 0.2 sp. nov. 3 63 X X X 5.9 1.6 x 1.0 1.1 2.2 drokel1sbergel1sis 63 X X X 5.9 2A x 0.9 1.2 2.0 0.9 x 0.3 0.9 x 0.3 mhra 64 X X X 8,4 2.7 x I.i 0.7 L7 0.8 x 0.3 filiformis subsp· fi/ijiJr/llis 6 3. 64 X X X 6 0 2.3 x 0.5 Ll 2.2 2.2 0.8 x 0.3 fili/armis subsp. nov. 66 X X X 6.5 2.5 x 0.6 1.2 0.8 L7 0.8 x 0.3 sp. nov. 4 C X X X 6.6 2.2xO.7 8,4 1.4 2.1 1.0 x OA falei/olill C X X X 2.5 x 0.7 2.1 x 1.1 0.7 IA 0.9 x OA Flt/garis 0 X X X 6.2

OJ * From 6redcnkamp & Van Wyk (2001). S X. character present. ::r ~ ~ . ."" j" N 8

Bothalia 31,2 (200 1) 225

FIGURE S.-LM photographs of P. ericoides, Bredenkamp 956, with fleshy fi1Jit, and P vulgaris, Bredenkamp 951, with dry fi1lit, showing structure of receptacle and base of ovary. A-D, tis at different levels of receptacle. A-C, P. vulgaris: A, departure of eight traces from central stele; B, carpel­ lary bundles ananged in ring, fused stamen and sepal traces in peripheral position; C, differentiation of cortex tissue into inner epidermis ofhypan­ thium and outer epidermis of ovary waU , separation of dorsal carpeUary bundle. D, comparison to similar stage in P. ericoides. E-H, tis at base of ovary showing vasculation of ovary wall, funiculus and chalaza of pendulous ovule: E, P. ericoides, ovary wall independent of hypantbium, crupellary bundles in a single whorl; F, P. vulgaris, young bud, ovary wall loosely adhering to hypantbium distally; G, P. ericoides, one cell of functional macrospore; H, comparison to similar stage in P. vulgaris, reduction of dorsal carpellary bundle. c, carpellary bundle; cc, commissur­ al carpeUary bundles; cs-pst, fused commissural sepal and antipetalous stamen bundles; d, area of tissue differentiation; dc, dorsal carpellary bun­ dle; f, funiculus; fm, functional macrospore; h, hypantbium; ieh, inner epidermis of hypantbium; ieow, inner epidermis of ovary wall; ii, inner integument; mc, median carpellary bundle; n, nucellus; 0 , ovule; oeow, outer epidermis of ovary wall; oi, outer integument; ow, ovary wall; p, placenta; s-sst, fused sepal midrib and antisepalous stamen bundJe; t, trichome; vh, vascular bundle of hypantbium. Scale bars: A- H, 100 }1m. 99

226 Bothalia 31,2 (2001)

FIGURE 6.-LM photographs of P. ericoides, Bredenkamp 956, with fleshy fruit, and P. vulgaris, Bredenkamp 951, with dry fruit, showing structure of ovary and ovule. A, B, Us in median position of ovary: A, P. ericoides, embryo sac and two cells of functional macrospore; B, higher magnification of similar stage in P. vulgaris. C, D, Us at top of ovary: C, P. ericoides, placenta, obturator and micropyle originating from inner integument; D, comparison of similar stage in P. vulgaris. Abbreviations as in Figure 5. es, embryo sac; ieii, inner epidermis of inner integument; ieoi, inner epidermis of outer integument; m, mesophyll; mi, micropyle; ob, obturator; oeii, outer epidelmis of inner integument; oeoi, outer epidermis of outer integument. Scale bars: B, 10 flm; A, C, D, 100 flm.

show that Meisner (18S7) regards the floral envelope as Hypanthium and androecial position perigynous and hypocrateriform, implying a cup-shaped receptacle or hypanthium. Gilg (1891, 1894) describes Owing to reduction of the receptacle, the hypanthium the floral envelope as a cylindric receptacle which is in Passerina is here interpreted as being formed by the articulate in the upper half, and Endlicher (1837, 1847), fused calyx and androecium only. The vascular tissue of Leandri (1930) and Dahlgren & Thome (1984) regard the hypanthium constitutes the fused sepal and stamen the floral arrangement as perigynous. The present study traces (Heinig 19S1), separating from the central stele in indicates that the receptacle (in tis) is very short (Figure a single whorl and forming a peripheral ring of eight vas­ SA, B) and definitely not cup-shaped. This is evident cular bundles (Figure SA, B), which persist throughout from the arrangement of peripheral cortex cells in rows, the length of the hypanthium. A similar pattern of fusion followed by differentiation into the inner epidermis of and distribution of vascular tissue has been reported by the hypanthium and the outer epidermis of the ovary wall Heinig (19S1) and for the genus Lachnaea (= Cryptade­ (Figure SD), finally by the separation of the hypanthium nia) by Beyers (1992) & Beyers & Van der Walt (199S). (including the vascular bundles differentiated from the In Passerina the central stele differentiates into carpellary stele) from the ovary wall and the presence of trichomes bundles after the separation of the fused sepal and stamen in the space subsequently formed (Figure SD-F). traces (Figure SC-E). It can therefore be concluded that 100

Bothalia 31,2 (2001) 227

FIGURE 7.- LM photographs of P. ericoides, Bredenkamp 956, with fleshy fruit, and P vulgaris, Bredenkamp 951 , with dry fruit , showing structure of micropyle and style. A, P. ericoides, obturator, pollen tube penetrating micropyle, opening of outer integument towards micropyle. B, similar stage in P. vulgaris, style departing laterally. C, D, tis of style. C, four-lobed style in P. ericoides. D, elliptic style in P. vulgaris. Abbreviations as in Figure 5. ct, conducting tissue ; mi, micropyle; ob, obturator; pt, pollen tube; st, style. Scale bars: C, D, 10 Jlm; A, B, 100 Jlm.

the segments of the floral envelope and the androecium indicates a reduction in tissue and a possible advanced arise below the gynoecium, the floral arrangement being state. This is indeed the case in Passerina. hypogynous and the ovary superior (Weberling 1989). A study of petaloid scales in Thymelaeaceae has added further evidence to the interpretation of the floral Identity offloral envelope envelope. These scales have been regarded by various authors as perigynous scales or glands, perigynous nec­ From Wikstrom (1818) to Takhtajan (1997) (Tables taries, petals or staminodes, petaloid scales and petaloid 1; 2) the floral envelope in Thymelaeaceae and, in many appendages (Tables 1; 2). Heinig (1951) is convinced cases Passerina, was variously interpreted as an in­ that the morphology and vascularization of the petaloid fundibular corolla, hypocrateriforrn hypanthium, infun­ scales resemble that of stipules, an opinion shared by dibular peri gone, perianth, cylindric receptacle, floral Rao & Dahlgren (1969) on the floral anatomy and rela­ tube formed by coalescence of four external whorls, tionships of the Oliniaceae. In their floral description of perianth tube, or a hypanthium. Heinig (1951) supports OUnia, Dahlgren & Van Wyk (1988) consider the the interpretation of Leandri (1930) and Domke (1934) petaloid scales as true petals. Heinig (1951) concludes of the floral tube as appendicular in origin, composed that the Thymelaeaceae is apetalous. In Passerina there of the fused bases of the sepals and adherent stamen are no petaloid scales or corolla (Tables 1; 2). Our filaments, also pointing out that the sepal is with few results have shown the separation of antipetalous as well exceptions a three-trace organ. Bunniger (1972) is of the as antisepalous stamens, but petaloid scales or even ves­ opinion that the hypanthiums in families of the Myrtales tiges of them were not observed. We therefore regard and Thymelaeales have a similar origin. Our results Passerina as truly apetalous. The complete reduction of show eight vascular bundles running along the length of the corolla or the absence of petaloid scales can be the hypanthium and separating into sepal and stamen regarded as an advanced state in the Thymelaeaceae; it bundles, each sepal lobe eventually with three vascular could also be interpreted as part of the anemophilous bundles (Figure 8A, B). We regard the floral envelope as syndrome displayed by the genus. Based on both the a hypanthium (fused calyx and androecium), differen­ pattern of the vascular tissue and the absence of petaloid tiating into four petaloid sepals and a diplostemonous scales, we consider the floral envelope in Passerina as a androecium, arising from the hypanthium rim at the hypanthium consisting of the fused calyx and androecium, separation of the sepals. A very short receptacle (Heinig differentiating into four sepals and the diplostemonous 1951), which does not contribute to the hypanthium, stamens.

101

N TABLE 8.-Specialized characters of the floral envelope in P(IHerilia. correlated with leaf anatomy N oc

Fragmentation of Hypanthiunl Sepals Taxon Leaf struc ~ypanth . aftcr fruiting tural type at neck Iat circumf. neck I indumcnlum indumentum \ inner 10bC:\ \ shape of lobes base of ovary length* at ovary I at neck outer lobc surface I inner lobe surface I x w* outer & inner outer I inner adaxial -I' abaxial adaxial I abaxial

sp. nov. I A x 1.2 tomentose tomentose glabrous apex setose glahrous glahrous 1.9 x 1.2 cymbiform widely ovate ericoides B3 x 0.6 strigosc strigose puberulent glabrous puberulent glabrous 1.6 x 2.0 concave, wide­ ly obovate rigidu B3, B6, x 0.8 glabrous tomentose midrib glabrous tomentose glabrous 1.8 x 0.9 cymbiforrn obovate 02 tomentose plileaceu B2, B3. x OJ gl~brous glabrous glabrous glabrous glabrous glabrous 1.8 x 1.5 concave. ellip­ B4,02 tic or subrotund sp. nov. 2 B3 x 0.7 tomentosc tomentose apex tomen­ apex setose glabrous apex sctose 1.8 x 1.0 concave, concave, obo­ tose oblong vate golpinii B2, B3 x 1.0 pubescent pubescent glabrous glabrous apex margin glabrous 2.4 x 1.6 concave, obovate tomentosc elliptic glomera/a subsp. glomera/a B2, B3 , x 0.8 glabrous tomentose scantily glabrous scantily glabrous 2.3 x 0.8 cymbiforrn oblong-elliptic B5 pubescent pube

Bothalia 31,2 (2001) 229

FIGURE 8.-LM photographs of P. ericoides, Bredenkamp 956, with fleshy fruit, and P vulgaris, Bredenkamp 951, with dry fruit, showing structure of androecium. A-D, serial tis of sepals and two anther whorls. A-C, P. ericoides: A, each of fused commissural sepal and antipetalous stamen bundles splitting into two sepal laterals and one antipetalous stamen bundle; B, fused sepal midrib and antisepa­ lous stamen bundle, splitting into sepal midrib and antisepalous stamen bundle; C, final stage in separation of antisepalous stamen bun­ dles. D, P. vulgaris, tis through both anther whorls. Abbreviations as in Figure S. pst, antipetalous stamen bundle; s, sepal midrib bun­ dle; sl, sepal lateral bundle; sm, stigma; sp, sepal; sst, antisepalous stamen bundle. Scale bars: A-D, lOO J-lm.

Fragmentation of hypanthium 1992; Beyers & Van der Walt 1995). We hypothesize that the plane of circumscission possibly indicates a differ­ The fruit in Passerina is enveloped by a persistent, ence in tissue composition between the basal and upper loosely attached hypanthium. Bentham & Hooker (1880), portions of the hypanthium and that this articulation can Gilg (1891, 1894), Domke (1934) and Melchior (1964) be of morphological importance in the Thymelaeaceae. mention that the hypanthium is articulated above the ovary. The basal portion of the floral tube below the plane of cir­ We found no definite articulation point in the hypanthium cumscission possibly indicates the inclusion of receptacle neck (narrowed part of hypanthium between apex of ovary tissue in the hypanthium, whereas the upper portion con­ and sepals) in Passerina, possibly because of the absence sists of calyx and androecium tissue only (accepting the of receptacle tissue and the appendicular nature of the apetalous state). An altemative interpretation, offered by hypanthium. The base of the neck fragmented as a result of one of the referees of this paper, regards the vasculature desiccation and torsification of cells (Figure 2E). In some as a prerequisite to decide whether one is dealing with a species, fragmentation of the hypanthium takes place over hypanthium (appendicular in origin) or a receptacle (axial the widest circumference of the fruit, shedding the frag­ in origin), The significance of an articulation indicating a mented distal part of the hypanthium, sepals and androeci­ distinction between parts of the hypanthium of different urn (Figure ID). A strong association was found between derivation, should be further investigated. flowers with a short hypanthium neck and fragmentation of the hypanthium over the widest part of the fruit, and also Androecium between flowers with a long hypanthium neck and frag­ mentation at the base of the neck (Table 8). In his description of Thymelaeaceae, Peterson (1978) describes the stamens as twice as many or as many as the A plane of circumscission, dividing the floral tube into sepals (rarely reduced to two or one), in one or two whorls, a basal and upper portion, is clearly illustrated in Gnidia the outer whorl antisepalous. In the subfamily Aqui­ and Struthiola (Peterson 1978) and Lachnaea (Beyers lmioideae (Heinig 1951), stamens are of approximately 103

230 Bothalia 31,2 (2001)

FIGURE 9.-LM photographs of P. ericoides, Bredenkamp 956 and P. vuLgaris, Bredenkamp 951, showing structure of anther locu lae in tis: A, P. vuLgaris, 4-locular anther; B, P. ericoides, cell wall thickenings of endothecium rib-like; C, P. vuLgaris, rupturing of partition between loculi; D, P. ericaides, rupturing of outer walls of thecae, extrorse. e, epidermis; en , endothecium; po, pollen. Scale bars: B, C, 10 flm; A, D, IOOflm. equal length and the traces supplying them separate from separation higher up in the floral tube, e.g. at the top of the sepal traces, practically in a single whorl. The the ovary in Dirca occidentalis and below the origin of Thymelaeoideae is characterized by stamens arising as the sepals in Gnidia subulata (Heinig 1951). In Passe­ two distinct whorls at two distinct levels in the floral tube. rina the separation of the antipetalous stamen traces The dimorphic diplostemonous nature of stamens in takes place at the origin of the sepals (Figure 8A, B) and Passerina (Thymelaeoideae), in which the antipetalous separation of the antisepalous traces slightly higher up stamens are shorter than the antisepalous ones, has been (Figure 8e), indicating what appears to be a phylogeneti­ confmned by our observations (Figure SA-C). According cally advanced tendency. to Heinig (1951) evolution within the androecium has been from po]ystemony to diplostemony to dimorphic Pollen diplostemony, indicating the advanced state of the androe­ cium in Passerina. Peterson (1978) describes the anthers Pollen grains of most members of Thymelaeaceae are as usually introrse, rarely extrorse. Species of Passerina monads, spheroid and pantoporate, characterized by a have large, exserted, extrorse anthers, clearly an adapta­ typical croton pattern, comprising rings of more or less tion to the anemophilous syndrome of the genus. trihedral sexine units mounted on an underlying reticulum of circular muri (Bredenkamp & Van Wyk 1996). In Separation offused sepal and stamen traces Passerina, the basal reticulum, as in the typical croton pattern, is no longer discernible as it is replaced by a sec­ Heinig (1951) is of the opinion that a foreshortening ondary reticulum derived from fused sexine. The supra­ of the floral axis has resulted in a fusion of the calyx and tectal subunits are fused completely to form a continuous androecium and that progressive stages of adnation can reticulum which replaces the underlying reticulum. The be observed in the family. In all species the antipetalous reticulum in Passerina is therefore secondary in origin stamen traces are fused to the comrnisural sepal traces and considered phylogenetically advanced. This modifi­ and the antisepalous stamen traces to the sepal midrib cation of the crotonoid pattern is probably also of func­ traces at their point of origin from the stele. In the tional significance as pollen in Passerina is adapted to Aquilarioideae, in Octolepis dinklagei, they become anemophily. separated in the receptacle or, in other cases, low down in the floral tube. Except for Gnidia splendens (= Lasio­ Disc siphon splendens), in which the separation of the antipetalous and antisepalous stamen traces takes place In Aquilarioideae a hypogynous disc is generally in the receptacle, Thymelaeoideae is characterized by absent, but is almost always present in Thymelaeoideae 104

Bothalia 31,2 (2001) 231

c D

FIGURE 1O.-LM photographs of P. ericoides, Bredenkamp 956, with fle shy fruit, and P vulgaris, Bredenkamp 951, with dry fruit, showing structure of young fruit and developing seed in tis. A, B, P. ericoides: A, pericarp fleshy, atrophy of outer integument; B, higher mag­ nification, outer epidermis of inner integument palisade-like, mesophyll, inner epidermis of inner integument tanniniferous. C, D, P. vulgaris: C, palisade-like exotegmen di scel11able in tegmen. D, tegmen black and lignified, crassinucellate, endosperm initially nuclear. Abbreviations as in Figure 5. ens, endosperm; es, embryo sac ; ieii, inner epidermis of inner integument; m, mesophyll; oeii, outer epidermis of inner integument; pc, pericarp; te, tegmen. Scale bars: A-D, 100 11m.

(Heinig 1951; Peterson 1978). Possibly because of a whorl a reduction series may be observed, ranging from reduction of tissue, no disc was observed in Passerina, a four- or more-carpellate members in the Aquilarioideae state confirmed by Bunniger (1972). to the two-carpellate members of the Thymelaeoideae in which one carpel is markedly contracted, thus a Ovary pseudomonomerous ovary. According to Heinig (1951) the ovules have been reduced to one per locule in the The ovary in Passerina was described as unilocular entire family. In Passerina, at the base of the ovary, the up to the time of Domke (1934) (Table 1). The pseudo­ dorsal and median carpellary bundles initially separate monomerous state is mentioned by Eckardt (1937), from the central stele (Figure 5C, D). After differentia­ Melchior (1964), Dahlgren & Thorne (1984) and tion has taken place, the dorsal, median and commissur­ Cronquist (1988) (Table 2), while most authors agree al carpellary bundles (Figure 5E) can be distinguished. that ovules are solitary and pendulous. According to Because of a redistribution of carpellary bundles, the sin­ Takhtajan (1997) the ovary in Thymelaeoideae consists gle dorsal carpellary bundle is arranged at the opposite of two carpels, it is monolocular and the ovule is solitary. side of the horseshoe-shaped median and corrunissural Heinig (1951) is convinced that within the carpellary carpellary bundles (Figure 5G). In Dirca palustris, 105

232 Bothal ia 31,2 (200 I )

Heinig (195 I) has illustrated the authenticity of the a specialized adaptation. A reduction in tissue from a drupe pseudomonomerous ovary by the presence of two dorsal to a membranous I-seeded berry or an achene can be illus­ carpellary bundles, one in the fertile carpel and one in the trated in Thymelaeaceae and therefore the fru it in second. reduced, sterile carpel positioned between two Passerina could be considered advanced within the family. groups of commissural carpelJary bundles. Bunniger We agree with Domke (1934) that P. ericoides (Figures IF; (1972) showed the presence of two carpels in the flower lOA) and P. rigida are characterized by a fles hy I -seeded primordia of P. fili/onnis. In the present study, which berry, while all the other species have an achene (Figure included the young bud stage of P. vulgaris (Figure 5F), 2F; Table 7). The achene remains enveloped in the rem­ no indication of a second carpel or a second dorsal nants of the papyraceous hypanthium, nestled adaxially in carpeIJary bundle was found, possibly because of reduc­ the tomentum of the concave. persistent cymbiform bracts. tion and fusion processes, which had already taken place in the formation of the young ovary, consequently we Seed consider the ovary of Passerina as pseudomonomerous. The existing confusion concerning the state of the Ovule type and position tegmen in Passerina is a reflection of the total lack of information of this aspect in descriptions of the group by Domke (1934) describes the ovule in Thymelaeaceae various authors (Tables I; 2). Meisner (1857) describes a as pendulous, anatropous, wi th a ventral fu niculus, ex­ crustaceous pericarp and Domke (1934) a black, crusta­ ceptionally hemi-anatropous or orthotropous. indicating ceous testa. The ovule in Passerina is bitegmic (Figures a phylogenetic tendency. Our study clearly indicates a 6; 7; 10). Comer (1976) refers to outer integument (oi) pendulous, anatropous ovule in Passerina (Figure 6C, and inner integument (ii), the product of the outer integu­ D). The funiculus is ventral and has been sectioned from ment becoming the testa and that of the inner integu­ the base of the ovule (Figures 5E-H; 6A) to the point of ment, the tegmen. In Passerina the outer integument dis­ attachment with the placenta (Figure 6C, D). Close to the integrates, whereas the inner integument remains (Figure embryo sac the micropyle isa trilete opening fo rmed by lOA), its outer epidermis becoming palisade-like (Figure the inner integument (Figures 6C, D; 7 A), facing ]OA, B). Hence, the seeds of Passerina are exotegmic upward. Towards the micropyle, the outer and inner with a palisade, a state common to the family (Comer integuments become horseshoe-shaped (in tis), resulting (976). In Passerina the tegmen is black and lignified, in the micropyle being directed towards the elongated and in tis, still portraying its origin from the palisade-like obturator cells, located at the base of the style (Figure epidermis (Figure IOC, D). 7B). Based on these results, we agree with Dahlgren ( 1975b), who regards the ovule as pendulous and Floral and fruit morphology at species level (Thbles 7; 8) epitropous. In the present study, specific results in leaf anatomy Obturator indicating the arrangement of taxa in Passerilla (Bredenkamp & Van Wyk 2001), are associated with flo­ Davis ( 1966) defines an obturator as any structure asso­ ral morphological structures as well as fru it and seed ciated with directing the growing pollen tube towards the types. In Table 7, leaf structural types A and especially micropyle, but elongated cells extending from the base of B3 are associated with four species that have smaller, the style to the micropyle are considered exclusive to the yellow, membraneous flowers (up to 5.3 mm long). Leaf Thymelaeaceae. In Passerina such elongated obturator structural types B4, B5, B6, C and D are associated with cells can be clearly seen at the level of the placenta, at the most species having larger, yellow-pink, papyraceous departure of the fun iculus, touching on the inner integu­ flowers (up to 8.4 mm long). The same tendency in the ment (Figures 6C; 7A) and finally these cells extend from length of the neck, and the size of the inner sepals is the base of the style entering the micropyle (Figure 7B). shown in Table 8.

Fruit Fnlit

Most authors (Tables I; 2) agree that the fruit in Fleshy fru it in P. ericoides (Figure 1D , F) and P. rigida Thymelaeaceae is indehiscent. In Passerina, Wikstrom is possibly correlated with the moist maritime climate of (1818), Meisner ( 1857) and Endlicher ( 1837, 1847) con­ the coastal habitat of these species; it is possibly also an sider the fruit as a nut or a nutlet. Domke (1 934) concludes adaptation to bird dispersal. Both species occur in the that the fruit of Dais, Gnidia, Lachnaea (= Cryptadenia) Western Cape, and the range of P. rigida extends along and many taxa of Passerina can be defmed as an achene, the coast to St Lucia. P. ericoides has red benies and and that of P. ericoides as a berry. Dahlgren (J975b) con­ P. rigida has yeUow berries. All other species are char­ siders the fruil of Thymelaeaceae as a nut or drupe, Peter­ acterized by achenes and are adapted to drier habitats, son (1978) regards it as a berry, a nut, a drupe or a loculi­ from mountainous areas along the Great Escarpment to cidal capsule and Takhtajan (1 997) describes them as nut­ the arid Karoo. In Pm'serino, each achene is enveloped like, baccate or drupaceous. A relevant family character, by papyraceous remnants of the fragmented hypantllium Ihat the outer integument of the ovule disintegrates and the and enclosed within the tomentum of an enlarged bract inner integument becomes palisade-like and hardens to (Figure 2D, E), which often takes on a rounded shape form a seed coat or tegmen (Figure ] OA-D), is illustrated and turns yellow, red or brownish. by the present study. Structurally the dry, membranous fruit in Passerina conforms to an achene (Spuyt 1994) and the The fruits of Passerino clearly illustrate the phenome­ reduction in pericarp tissue can probably be considered as non of transfer of function from protection to dispersal 106 Bothalia 31 .2 (200 I) 233

(Stebbins 1974). In P. ericoides and P. rigida, with fleshy Floral envelope fruit, the pericarp has the double function of protecting the ovules during early stages of development and disper­ The hypanthium and sepals in P. ericoides are char­ sal. The mature fruit enlarges beyond the bracts and is acterized by their coriaceous (almost fleshy) appearance protected by the coriaceous pericarp, while the patent and dull green to pinkish colour. The floral envelope in bract does not have a protective function (Figure 10). The P. rigida, P. paleacea, P. sp. nov. I and P. sp. nov. 2 is pericarp of the fruit is yellow or red when it is ready for pale yellowish and quite membranous. P. pendula is dis­ dispersal , probably by birds or small mammals (Richards tinguished by a pinkish floral envelope, with a membra­ 1986), and the dispersal unit is the berry. In all other nous texture. In all the other species the fl oral envelope species which are characterized by achenes, the protec­ is yellow-pink at anthesis, with a papyraceous texture. tion of the ovule is apparently transferred from the peri­ After pollination these flowers tum red and the hypan­ carp to the persistent bract. The bract enlarges around the thium and sepals become thinly papyraceous and dry. achene, protecting it in the woolly tomentum of the con­ cave adaxial surface (Figure 20). The mature fruit is For practical purposes the total length of the floral often still enveloped by the reddish, papyraceous rem­ envelope indicates flower size, and its taxonomic impor­ nants of the hypanthium. Both P. montana and P. glom­ tance is clearly illustrated by the general increase in size erata are characterized by subcapitulate inflorescences, from species )-20 (Table 7). P. rigida, P. paleacea and with proliferating growth more common in the latter P. sp. nov. 2 are characterized by small flowers, the species. In P. glomerata (growing in the arid Karoo) the length of the fl oral envelope 4.0--4.6 mm. In most other accompanying bract turns yellow and becomes more species it ranges from 5.3-7.3 mm long. P. rubra and P. patent when the fruit is mature; the achene is shed after falcifolia are characterized by large flowers, the floral abscission. The yellow colour is associated with senes­ envelope being 8.4 mm long. cence of the bracts and these structures are eventually shed, leaving conspicuous bract scars on the remaining, Specialized characters (Table 8) often terminal, woolly inflorescence axis. The unit of dis­ persal, in this case, is the achene, assisted by the patent a. Fragmentation of hypanthium after fruiting senescing bract. The achene falls to the ground where it could either be dispersed by ants or small mammals or During the fruiting phase, the persistent hypanthium germinate under favourable conditions. In P. montana fragments over the broadest part of the ovary, in eight of (growing along the Great Escarpment), the margin of the the 20 species. Except ill P. obtusifolia, the hypanthium fruiting bract turns red and it becomes more patent, of all these species is characterized by a short neck of exposing the achene enveloped in the beak-like, reddish, 0.3-1.0 mm. Anatomically their leaves also correlate papyraceous hypanthium, which fragments at the neck with leaf structural type B (Bredenkamp & Van Wyk base. Perhaps birds, attracted by the red colour (Richards 2001). When fragmentation occurs at the neck base, the 1986), feed on the exposed achenes. Leafless, terminal, hypanthium usually has a long neck length of mostly woolly branchlets, with terminal scars are a conspicuous 1.3-3.0 rom, and such species are associated with leaf feature of the plant after fruiting and it therefore also structural types A, B, C and O. seems possible that the subterminal capitulum with sever­ al achenes is broken off as a unit. In this case the disper­ b. Indu mentum of hypanthium sal unit could be the achene or the achene accompanied by the bract or perhaps even the entire subcapitulum. In certain species of the genus Lacllllaea (Beyers 1992; There is a need for further observations on seed dispersal Beyers & Van der Walt 1995), different trichome types are in the field to test some of these suggestions. found below and above the articulation plane in the hypan­ thium. This state could possibly also be present in other Filament length genera of the Thymelaeaceae. In Passerina the trichome type remains constant over the length of tbe hypanthium, For a comparison of fi lament length, it is easier to possibly because there is no articulation plane in the measure the antisepalous filaments as they are ± twice hypanthium. As Passerina is distributed over a wide range the length of the antipetalous ones. Both P. sp. nov. 1 and of habitats, the density of the indumentum has been impor­ P. sp. nov. 2 have short antisepaJous filaments of ± 1.2 tant in the distinction of certain species (Table 8). P. eri­ mm, corresponding to their small flowers. P. paludosa, coides is distinguished by the strigose indumentum over P. flIiformis and P. f alcifolia have exserted stamens the length of the hypanthi um, whereas the indumenlum of because of their long (2.1-2.4 mm) filaments. the neck is strigose in P. paludosa. A completely glabrous hypanthium is characteristic of P. paleacea. In 12 of the Anthers species, the hypanthium surrounding the ovary is glabrous, scantily tomentose or tomentose at the apex, Conspicuous differences in anther size have been with the neck scantily tomentose or tomentose. In P. sp. noticed among the species; this has also been reported by nov. I, P. sp. nov. 2 and P. flliformis subsp. filifonnis the Thoday (1 924). In relation to fl ower size, most species hypanthium is tomentose over its entire length . In have large exserted anthers between 0.7(-0.9) x P. galpinii the whole of the hypanthium is pubescent, 0. 3(-0.7) mm, possibly an anemophilous adaptation. P. whereas in P. rubra only the neck is pubescent and the rest ericoides has ± elliptic anthers (Figure I), whereas those of the hypanthium is glabrous. of P. drakensbergensi!J' are narrowly oblong, 0.9 x 0.3 mm. The largest anthers of 1.0 x 0.5 rum are found in P. A strong correlation was found between the indumen­ Oblusifolia and P. falcifolia. tum of the lower hypanthium and of the bract. There is a 107 234 Bothalia 31,2 (2001)

te ndency for species characterized by a glabrous hypan­ es. Thus Dahlgren (1980) placed the Thymelaeaceae in thium base (Table 8) to have a protective bract with a the superorder Malviflorae (= Dilleniiflorae), Cronquist very hairy adaxial surface (Table 5), whereas species (1981, 1988) placed it in the subclass Rosidae, Thorne with a hairy hypanthium base have a less hairy to almost (I 992a, 1992b) in the superorder Malvanae and glabrous adaxial bract surface. When the hypanthium Takhtajan (1997) in the subclass DiIIeniidae (Table 2). neck is not covered by a bract, it tends to be hairy in varying degrees. Only P. paleacea has a completely Heinig (1951 ) discussed the relationships of the glabrous hypanthium. In this species the entire bypanthi­ Thymelaeaceae with the Myrtales, Saxifragaceae, urn is completely covered by the hairy adaxial surface of Lythraceae, Gonystylaceae and Malvales on the basis of the bract because of the very short hypanthium neck. floral morphology and concluded that a polyphyletic ori­ This tendency shows that the function of protection of gin of the Thymelaeaceae could be sought in both the the ovule is partly transferred from the hypanthium to the Flacourtiaceae and Tiliaceae. hairy bract (Stebbins 1974). Cronquist (1968, 1981 , 1988) considered the c. Indumentum of sepals Thymelaeaceae as completely at home in the Myrtales (Rosidae) on account of the strongly perigynous Thoday (1924) uses the character 'outer sepals beard­ polypetalous to apetalous flowers, internal phloem, ves­ ed behind the apex' in his key to distinguish between tured pits and obturator. However, he admited a possible species. The abaxial surfaces of the outer and inner lobes relationship with other families, based on the of seven species are setose at the apex only and the adax­ pseudomonomerous ovary and crotonoid pollen. Dahl­ ial surfaces range from glabrous to variously hairy (Table gren (I975a, b) placed the superorder Thymelaeanae 8). In P. comosa abaxial surfaces of both outer and inner between the Dillenianae (DilIeniales, Cistales, Malvales, lobes are tomentose and adaxial surfaces are glabrous, Urticales, Euphorbiales) and the Myrtanae. Within the P. drakenshergensis is similar except for the tomentose superorder Malviflorae, Dahlgren (1980) recognized a adaxial surface of the inner lobes. All sepals are com­ close affinity between the Malvales and Euphorbiales, pletely glabrous in P. paleacea. The indumentum of the and indicated a strong relationship with the Urticales and sepals varies infraspecificalJy and should, however, be the Thymelaeales, but a phylogenetic affi nity between used with discretion to distinguish between groups of the Malviflorae and Myrtiflorae was not supported. The species. inclusion of the Thymelaeaceae in the Myrtales was reviewed by Dahlgren & Thome (1984). They argued d. Size and shape of sepals that anatomically, most members of the fami ly possess Myrtalean characters. On the other hand, embryological, The size of the inner sepals is of taxonomic impor­ chemical and palynological evidence strongly indicates tance (Table 8). P. sp. nov. I, P. ericoides, P. rigida, P. an affinity with the Mal viflorae. Thorne ( 1992a) accept­ paleacea and P. sp. nov. 2 are characterized by shorter ed the superorder Malvanae, but incl uded the Thyme­ and broader inner sepals, varying from widely ovate, laeaceae in the order Euphorbiales. Takhtajan (1 969) obovate, widely obovate to subrotund. AU other species considered the Thymelaeales to have a common origin have longer, narrower inner sepals, the shape varying, with the EuphorbiaJes and Malvales, all arising from a with one exception, from narrowly oblong, oblong, Flacourtiaceae-type ancestor, and Takhtajan ( 1997) oblong-elliptic, elliptic, to obovate. P. drakensbergensis placed the Thymelaeales in the superorder Euphor­ is distinguished by lanceolate sepals arranged in the bianae, with the Gonystylaceae as the only other family shape of a cross. in the order.

Less important taxonomic characters Palynological evidence indicates that the very distinc­ tive pollen in Thymelaeaceae is totally different from Ovary size (Table 7) has been considered less impor­ that of any Myrtales and si milar to that of most tant, as it is difficult to measure all ovaries at the same Euphorbiaceae. Archangelsky (1 971 ) concl uded that developmental phase. In Passerina the size of the ovary both the Euphorbiales and the Thymelaeales belong to increases markedly after anthesis and the enlarged ovary, the subclass Dilleniidae and originated from ancestral enveloped by the persistent hypanthium. can already be lines of the Dilleniales -? Violales -? Malvales. In a observed in older fl owers, rapidly followed by matura­ paper on palynology of Passerina, Bredenkamp & Van tion of the fruit. Wyk (1 996) supported Dahlgren (1 980) in placing the Thymelaeales in the Malviflorae (= Dilleni iflorae). Taxonomic relationships In Passerina, the structure of the integuments sur­ Up to 1930, pri ority was given to the definition of rounding the ovule provides taxonomically useful subordinal or subfamilial taxa in Thymelaeaceae, based embryological evidence. We have hown the disintigra­ mainly on fl oral morphology (Table I). Applying both tion of the outer integument and the differentiation of the morphological and anatomical evidence, Domke (1934) jnner integument into a palisade-like outer epidermis, a proposed a satisfactory subfamilial classification system mesophyllayer and an inner epidermis (Figure lOA, B). and envisages a phylogenetic relationship between the It is also clear that the mechanical part of the seed coat is Thymelaeaceae, Malvaceae and Euphorbiaceae. Modern derived from the palisade-like outer epidermis, hence it techniques have enabled taxonomists to find relation­ is an exotegmen (Figure lOC, D). Comer (1976) distin­ ships between families and to arrange them into higher guished the Euphorbia les-Malvales-Thymelaeales­ hierarchies, with ranks such as superorders or subclass- Tiliales (Malvalean complex) on the basis of the exo­ 108 BOlhalia 3 1,2 (2001 ) 235

tegmic palisade. He dismissed the derivation of the Considering all the characters mentioned, Passerina is Malvalean complex from the Dilleniales (endotestal considered highly advanced in relation to other genera in seeds) or Violales and looked towards the Myristicaceae Thymelaeoideae. (MagnoJiales-Ranales) for the origin of the Malvalean seed. Systematic value

Recent evidence from molecular phylogeny (Table 3) Family level should be interpreted in context wi th the evidence from other botanical fields. The primary focus of The The exotegmic palisade and the distinctive obturator Angiosperm Phylogeny Group (APG 1998) is on orders, are regarded as family characters. They form the basis of with a secondary focus on families of flowering plants. Corner's (1976) Euphorbiales-Malvales-Thymelaeales­ Above the ordinal level , ranks are defined as subgroups, Tiliales complex, clades or supraordinal subgroups. Magallon et al. (1999) attempted to compare these groups to the existing systems Subfamily level mentioned in the preceding paragraphs. Both APG (1 998) and Magallon et aJ. (1999) recognized the Eudicots, a The Thymelaeoideae is distinguished on the basis of group characterized by tricolpate poilen, as well as the the calyx tube (hypanthium in the present study ), supra ordinal group Core Eudicots, supported by pen­ diplostemous androecium and pseudomonomerous ovary tamerous and isomerous flowers. Thymelaeaceae is desig­ (Domke 1934; Heinig 195 1). nated to the Rosid clade by most authors. APG (1998) placed the Thymelaeaceae in the subgroup Eurosids II and Tribal level order Malvales, whcrea:; all the other authors placed it in the Expanded Malvales. The Malvalean relationship of the On the basis of the secondary reticulum, unique to the Thymelaeaceae seems to be strongly supported by molec­ pollen of Passerina, Bredenkamp & Van Wyk (1996) ular phylogeny, as well as floral morphology, anatomy, raised the subtribe Passerininae to the tribe Passerineae. embryology and palynology. Genus level The EuphorbiaJes-Malvales-Thymelaeales relationship indicated by embryology and palynology is, however, The present study indicates the exserted, extrorse not supported by molecular data. APG (1998) placed the anthers and the anemophilous habit as unique to Passe­ Euphorbi aceae in the order Malpighiales in the subgroup rina. Euros ids I and Magallon et al. (1999) placed it in the Core Rosid Clade. Cronquist (1 968, 1981 , 1988) was Species level convinced that if the Thymelaeaceae is not placed in the Myrtales, it would stand next to it. Conti et af. (1996 ), Characters usef ul at species level are summarized in APG (1998), and Alverson et al. (1999) all regarded the Tables 7 and 8. Myrtales as a sister group of the Malvales or the Expand­ ed Malvales to which the Thymelaeaceae is designated. CONCLUSIONS

Speculations on phylogeny The evidence on floral morphology not only con­ firmed the identity of 20 species and four subspecies, but Within Thymelaeaceae, both Domke (1934) and also proved significant in the taxonomy of the genus. The Heinig (1 95 1) agreed that the subfamily ThymeJaeoideae status of the following taxa is confirmed by the present is phylogenetically more advanced than the Aquilari­ floral morphological study : P. burcheLlii Thoday, oideae. On the basi s of the advanced pollen, Bredenkamp P comosa C.H.Wright, P drakensbergensis Hilliard & & Van Wyk (1996) raised the subtribe Passerininae to the B.L.Burtt, P ericoides L., P falcifolia c.H.Wright, P.fil­ tribe Passerineae, a decision supported by the present ifonnis L. subsp. filifonnis, P. filifomlis L. subsp. nov., study, Although many of the following advanced charac­ P galpinii C.H.Wright, P glomerata Thunb. subsp. ter states are present in other genera of the Thyme­ glomerata, P. glomerata Thunb. subsp. nov., P montana Jaeoideae, these advanced character states are alJ found Thoday, P obtusifolia Thoday, P paleacea Wikstr., together in Passerina: receptacle reduced to a ± lenticu­ p, paludosa Thoday, P. pendula EckL & Zeyh " P. rigida lar structure; departure of the fused sepal and stamen Wikstr., P rubra C.H.Wright, P. vulgaris, P sp. nov. I, bundles before carpellary bundles; hypogynous floral P sp. nov. 2, P sp, nov. 3 and P. sp. nov. 4. arrangement; petal-like floral envelope comprising a hypanthium (fused calyx and anciroecium), differentiat­ For al most three centuries evidence from floral mor­ ing into four sepals and a diplostemonous androecium; phology has been basic to plant taxonomy and applied at separation of stamen bundles high up in hypanthiu m, at all hierachical levels, Our research on the flowers in formati on of sepals; exserted, extrorse anthers; anemo­ Passerina has produced new morphological and anatomi­ phiJous habit; secondary reticulum of pollen; complete cal evidence, especially as Heinig's classical study of floral absence of petals or petaloid scales; asymmetric devel­ morphology in Thymelaeaceae (1951) did not include opment of the style; superior, pseudomonomerous, uni­ Passerina. The present study has succeeded in resolving locular ovary; asymmetric attachment of ovule at top of the floral morphology in Passerina, as man y mistakes ovary; ventrally epitropous ovule; distincti ve obturator; have been perpetuated by previous authors. W conclude bitegmic ovule with exotegmic palisade; fruit a I-seeded that the flower in Passerina is a phylogenetically berry or an achene; seed with lignified, black exotegmen, advanced structure and consider the genus advanced

109 236 Bothalia 3 I ,2 (200 1)

cumscnptlOn variation, and relationships. Annals of Ihe within the Thymelaeoideae. Possible taxonomic relation­ ships of the Thymelaeaceae with the Malvales are strong­ Missouri Botanical Garden 7 I: 633-699. DAHLGREN, R.M .T. & VAN WYK, A.E. 1988. Structure and rela­ ly supported by the presentation of floral morphological, tionships of families endemic to or centred in southern Africa anatomical, embryological and palynological information Monographs in Systematic Botany 25: 1-94. to which this study has also contributed. FinalJy, all the DAVIS, G.L. 1966. Syslematic embryology oflhe angiospems. Wiley, above-mentioned evidence serves to form a firm taxo­ New York . DOMKE, W. 1934. Untersuchungen iiber die systemalische I.Ind geo­ nomic basis for future comparative studies, especially in graphische G/iederung der ThYlllelaeaceen . E. Schweizer­ the expanding molecular field. bart'sche Veriagsbuchhandlung (Erwin Nagele) G.M.B.H., Stuttgart. ECKARDT, I. 1937. 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